The present invention relates to methods for detecting, prognosing and staging cancers, in particular cancers of the gastrointestinal tract. The methods of the invention comprise detecting specific protein markers in a tissue of interest, wherein the detected levels thereof may be indicative of pre-cancerous or cancerous tissue, or the stage or prognosis of a cancer. Further provided are methods of treating cancer, and cancer detection kits.
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1. A method for detecting a colorectal pre-cancerous tissue in a subject, comprising the steps of:
(i) contacting in vivo a colorectal tissue with a pharmaceutical composition comprising a reagent suitable for detecting a combination of polypeptide markers; and
(ii) detecting in said colorectal tissue the presence of said combination, wherein said combination comprises:
NAMPT and OLFM4,
thereby detecting a colorectal pre-cancerous tissue in a subject.
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The present invention relates to methods for detecting, prognosing and staging cancers, in particular cancers of the gastrointestinal tract. The methods of the invention comprise detecting specific protein markers in a tissue of interest, wherein the detected levels thereof may be indicative of pre-cancerous or cancerous tissue, or the stage or prognosis of a cancer. Further provided are methods of using the markers for treating cancer, and cancer detection kits.
Colorectal cancer, also referred to as colon cancer or large bowel cancer, is a malignant neoplastic disease associated with tumors in the colon, rectum and appendix. With 655,000 deaths worldwide per year, it is the third most common form of cancer and the second leading cause of cancer-related death in the Western world.
Colorectal cancers originate in the colorectal epithelium and typically are not extensively vascularized (and therefore not invasive) during the early stages of development. The transition to a highly vascularized, invasive and ultimately metastatic cancer, which spreads throughout the body, commonly takes ten years or longer. If the cancer is detected prior to invasion, surgical removal of the cancerous tissue is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and black tarry stool. Generally, such symptoms are present only when the disease is well established, often after metastasis has occurred, and the prognosis for the patient is poor, even after surgical resection of the cancerous tissue. For example, patients diagnosed with early colon cancer generally have a much greater, five-year survival rate as compared to the survival rate for patients diagnosed with distant metastasized colon cancer. Accordingly, early detection of colorectal cancer is of critical importance for reducing its morbidity.
Diagnostic methods for colon cancer most frequently depend on direct visual inspection of the gastrointestinal (GI) tract. Endoscopy involves inspection with a miniaturized light source at a probe end of a coherent bundle fiber optic cable. Reflected light beam images are returned through the fiber optic cable for detection by an external digital camera and display on an external monitor or for recording on an external video recorder or both. While this technique allows for identification, removal, and biopsy of potentially cancerous growths such as polyps, its use is associated with certain disadvantages, such as being expensive, uncomfortable, inherently risky due to its invasive nature, and the inability to access some portions of the large intestine and most of the small intestine.
Swallowable endoscopy capsules containing miniaturized optical, digital camera and radio transmission systems have been subsequently developed along with complementary external monitoring systems for inspecting the GI tract. For example, the capsule marketed under the trade name PillCam® SB was initially approved by the U.S. Food and Drug Administration in 2001 for detection and diagnosis of disorders of the small intestine. U.S. Pat. No. 5,604,531 discloses an in vivo video camera system comprising a swallowable capsule. The transit of endoscopy capsules through the small intestine is dependent on peristalsis, meaning that some areas with lesions may be missed if the capsule is not retained in that area for a sufficient amount of time. Further, the endoscopy capsules in current use are not capable of identifying molecular markers, which may be early indicators of colorectal cancer, even prior to the development of pre-cancerous polyps.
U.S. Pat. No. 7,468,044 discloses a system and method for in vivo and in situ detection of body lumen conditions, such as in the GI tract. The system comprises an interaction chamber comprising an indicator; a light source for illuminating the interaction chamber; and an optical detector for detecting in vivo optical changes occurring in the interaction chamber upon reaction of the indicator with an endo-luminal sample.
U.S. Pat. No. 7,515,953 discloses a method for detecting fluorescence emitted by cells in a wall of a body lumen, such as an intestinal wall, the method comprising use of a swallowable capsule, the capsule comprising a light source and a fluorescent-labeled probe, which is released from a reservoir in the capsule. According to the disclosure, an electric field generated from an electrode in the capsule enhances uptake of the probe, and a detector in the capsule detects the fluorescent signal emitted. By determining the intensity and/or position in the lumen wall of the fluorescent signal, a drug for killing abnormal cells is released from a second reservoir in the capsule. According to the disclosure, the abnormal cells may be cancer cells, colon polyps or precancerous cells.
U.S. Patent Application Publication No. 2008/0146896 discloses a device, such as an autonomous capsule, for in vivo analysis which includes a reaction chamber to store a detecting reagent able to react with a sample collected in vivo; and optionally a labeled-substance chamber to store a labeled substance able to bind to a compound resulting from a reaction of the detecting reagent and the sample. According to the disclosure, the detecting reagent may be an antibody.
U.S. Patent Application Publication No. 2009/0216082 discloses a device system for in vivo detection of target molecules in an endo-luminal sample, and a method for in vivo magnetic immunoassay, which may be used for the detection of cancer in the gastrointestinal tract.
Yet other methods of colon cancer detection are based on detection of particular proteins or genes which are considered to be specifically or differentially expressed in colon cancer.
U.S. Pat. No. 7,507,541 discloses a method of detecting the presence of inter alia colon cancer that is based on determining the level of 36P6D5 protein expressed by cells in a test tissue sample from an individual, and comparing the level to that expressed in a corresponding normal tissue sample.
U.S. Pat. No. 7,501,242 discloses a method of detecting colon cancer that is based on detecting levels of expression of tyrosine threonine kinase (TTK) in a test sample, such as a colon sample, that are increased by at least two fold relative to the level of expression in a normal non-cancer sample of the same type.
U.S. Pat. No. 7,452,727 discloses a automatable method for identifying cancer cells and their precursor cells that is based on detecting at least two molecular markers, wherein the detection of each marker alone is not a reliable indicator of the presence of cancer cells and their precursor cells. According to the disclosure, the molecular markers may be selected from her2/neu, Ki67, p53, her2/neu, bcl-2, MN, mdm-2, EGF receptor, bcl-2 and p16.
U.S. Pat. No. 7,402,403 discloses a method for the detection of cancer or early neoplastic change that is based on detecting autoantibodies directed to tumor marker antigens in a sample of bodily fluids, wherein the tumor marker antigens are selected from MUC1, p53, c-erbB2, Ras, c-myc, BRCA1, BRCA2, PSA, APC and CA125.
U.S. Pat. No. 7,129,043 discloses a method of identifying a human subject having an increased risk of developing colon cancer that is based on detecting upregulation of the CLN3 gene.
U.S. Pat. No. 7,115,368 relates to a method of detecting epithelial cancer cells inter alia colon cancer that is based on detection of expression in a biological sample of pellino proteins.
U.S. Pat. No. 7,098,008 relates to a method for detection of cancer inter alia colon cancer that is based on detecting expression of melanoma antigen gene (MAGE).
U.S. Pat. No. 7,078,180 relates to a method of diagnosing a cancer inter alia colon cancer that is based on detection of a ZEB (zfh-1/delta EF1) polypeptide.
U.S. Pat. No. 6,949,339 relates to methods for detecting, diagnosing, monitoring, staging, and prognosticating colon cancers, based on detection of Colon Specific Genes.
U.S. Pat. No. 6,919,176 discloses a method of detecting cancer inter alia colon cancer that is based on detection of expression of specific G-protein coupled receptors.
There remains an unmet need for methods of early detection, prognosis and treatment of colon cancer.
The present invention provides methods of detecting cancer that are based on the qualitative or quantitative identification of particular proteins, also referred to herein as molecular markers. Further provided are methods of cancer prevention, prognosis and treatment.
Disclosed herein for the first time is a specific group of protein markers which are indicative of both pre-cancerous and cancerous lesions. Further disclosed herein for the first time is an additional specific group of protein markers which are primarily indicative of pre-cancerous lesions.
The invention is based in part, on the unexpected discovery that the level of expression within the gastrointestinal tract of certain proteins is significantly increased in both pre-cancerous and cancerous tissue relative to the level of expression of the same proteins in healthy tissue of the same type Surprisingly, the expression of these markers is elevated, even compared to healthy tissue bordering tumor growth. It has also been surprisingly found that the expression of yet other markers is significantly increased in early stage cancer, and significantly decreased in later stages of cancer
Without wishing to be bound by any particular theory or mechanism of action, the invention enables identification of individuals at risk of developing cancer, in particular colorectal cancer, even prior to observable histological changes in affected tissue. Since the methods of the invention are based on changes in protein expression patterns in cells, rather than later occurring pathological changes in tissue, a level of sensitivity is obtained that is greater by many orders of magnitude than current techniques of cancer detection. Thus, in the case of colorectal cancer, the invention provides a means of predicting the disease well in advance of the possibility of detecting potentially cancerous polyps by conventional endoscopic examination, the latter being the current yet inadequate standard for early detection.
The principles of the current invention are exemplified herein by quantitative mass spectroscopy analysis of healthy, pre-cancerous and cancerous tissue obtained from the gastrointestinal tract of patients during surgical excision of early stage (e.g. polyps) or more advanced stage tumors, which has resulted in the identification of a specific group of proteins, the expression of each of which is significantly increased in diseased colon tissue, as compared to healthy colon tissue. This group of proteins, which includes KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), has not been previously disclosed, either in part or as a whole, to be useful for detection of colorectal cancer at any stage of the disease.
It is to be specifically understood however, that the current method of the invention need not be limited to examination of colon tissue obtained by surgical means, nor should it be limited to detection and quantification of the subject molecular markers using mass spectrometry techniques. Rather, the invention may be advantageously practiced using for example immunological techniques and reagents for detection of the subject molecular markers, either in vivo or ex vivo. For example, labeled monoclonal antibodies can be used for in vivo detection and quantitation of such proteins in different tissue compartments and regions. The detection may be accomplished for example, using pharmaceutical compositions or endoscopy probes which incorporate specifically designed chemical, immunological or nucleic acid reagents. Advantageously, labeled reagents such as antibodies, which specifically interact with the subject molecular markers may be prepared as injectable or ingestible pharmaceutical compositions and following administration the interaction with their molecular targets may be externally monitored. Alternately or in addition, the invention may be practiced by analysis of biological samples obtained from a subject, such as biopsy tissue.
In a first aspect, the invention provides a method of detecting cancer in a subject, the method comprising: (i) detecting in a biological sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein, wherein detection of a level of said at least one protein in the biological sample which is significantly different from the reference level, is indicative of cancer in the subject.
Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In another aspect, the invention provides a method for determining the stage of a cancerous or pre-cancerous growth in a subject, the method comprising: (i) detecting in a test sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein; wherein the level detected in the test sample is indicative of the stage of the growth.
Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In another aspect, the invention provides a method for determining the prognosis of a cancerous disease in a subject, the method comprising: (i) detecting in a test sample from the subject at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), so as to determine the level of the at least one protein; and (ii) comparing the level determined in (i) to a reference level of the same at least one protein; wherein the level detected in the test sample is indicative of the prognosis of the cancerous disease.
Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the biological sample is selected from the group consisting of blood, serum, nipple aspirate fluid, lymph node aspirate, a biopsy sample, a tumor sample, a tissue sample, mucosal fluid, cervical wash, lacrimal duct fluid, urine, saliva, pleural effusion and sputum. Each of the aforementioned materials represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In currently preferred embodiments, the biological sample comprises a tissue sample. In currently preferred embodiments, the tissue sample comprises gastrointestinal tissue, particularly colorectal tissue or pre-cancerous tissue such as polyps. In currently preferred embodiments, the biological sample is selected from a tumor within the gastrointestinal tract, particularly a colorectal tumor. In particular embodiments, the gastrointestinal tissue is from an area or organ selected from the group consisting of the esophagus, the stomach, the small intestine, the large intestine (colon), the rectum, the appendix and a combination thereof.
In particular embodiments, the cancer being detected is selected from the group consisting of adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, fallopian tube cancer, gastric cancer, head and neck cancer, hepatic cancer, lung cancer including small cell lung cancer and non-small cell lung cancer, melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid and parathyroid cancer, renal cancer, sarcoma, thymoma, hematological malignancies and germ cell tumors. In a currently preferred embodiment, the cancer is colorectal cancer. Each of the aforementioned cancers represents a separate embodiment of the invention and may be used independently from any of the others.
In a particular embodiment, the at least one protein detected is indicative of a disorder selected from the group consisting of pre-cancerous polyps, early stage colorectal cancer and advanced stage colorectal cancer. In particular embodiments, a method of detecting pre-cancerous polyps or early stage colorectal cancer comprises detecting at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20); and further comprises detecting at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, a method of detecting pre-cancerous polyps or early stage colorectal cancer comprises detecting at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2); and further comprises detecting at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, a method of the invention comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20).
In particular embodiments, said reagent is suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, a method of the invention further comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the biological sample or test sample is obtained from the subject by a procedure selected from the group consisting of biopsy, flexible endoscopy, double balloon endoscopy and surgical colorectal re-sectioning.
In particular embodiments, the biological sample or test sample is assessed in vivo in the subject. In particular embodiments, the method comprises contacting a body tissue, cavity or fluid with at least one of a pharmaceutical composition and an endoscopy apparatus.
In particular embodiments, the method comprises contacting a body tissue, cavity or fluid with at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20)4. In particular embodiments, the body tissue, cavity or fluid is contacted with a reagent is suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the method further comprises contacting a body tissue, cavity or fluid with at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the method comprises administering a diagnostic pharmaceutical composition. In particular embodiments, the administering of the pharmaceutical composition is by a route selected from the group consisting of oral, parenteral, subcutaneous, intramuscular, intrathoracic and intraarticular.
In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one reagent suitable for detecting the level of at least one of the aforementioned proteins. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one reagent suitable for detecting the level of at least one of KIAA0152 (SEQ ID NO:1) and NAMPT (SEQ ID NO:2).
In particular embodiments, the reagent specifically interacts with at least one of the aforementioned proteins or with nucleic acid encoding at least one of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprises a multiplicity of reagents, wherein each reagent of the multiplicity specifically interacts with one distinct protein or with nucleic acid encoding one distinct protein or a fragment thereof. In particular embodiments, the reagent is selected from an antibody, an antibody mimetic and a nucleic acid. In particular embodiments, the antibody is selected from a monoclonal antibody, a humanized antibody, a single chain antibody, an antibody fragment and combinations thereof. In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise a multiplicity of antibody mimetics, wherein each antibody mimetic of the multiplicity specifically interacts with one of the aforementioned proteins.
In particular embodiments, the reagent comprises a detectable label, such as a fluorescent label, a radiolabel or an enzymatic label.
In particular embodiments, the pharmaceutical composition or the endoscopy apparatus comprise at least one least one nucleic acid, wherein the at least one nucleic acid is complementary to a nucleic acid encoding at least one of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the at least one nucleic acid comprises a multiplicity of nucleic acids, wherein each nucleic acid of the multiplicity is complementary to a nucleic acid encoding one distinct protein of the aforementioned proteins, or a fragment of said protein. In particular embodiments, the nucleic acid comprises a detectable label, such as a fluorescent label, a radiolabel or an enzymatic label.
In particular embodiments, the detecting in step (i) comprises use of an assay system. In particular embodiments, the assay system comprises an immunoassay, a nucleic acid hybridization assay, a binding assay, an array, a phage display library or combinations thereof. In particular embodiments, the array is a protein array, or a phage display library array. In particular embodiments, the assay system comprises at least one reagent suitable for detecting the level of at least one of the aforementioned proteins, as described herein. In particular embodiments, the assay system comprises a multiplicity of such reagents, for example antibodies, wherein each reagent in the multiplicity specifically interacts with one of the aforementioned proteins.
In particular embodiments, the detecting comprises use of an external monitoring system. In particular embodiments, the external monitoring system is configured to display the level of the at least one protein detected.
In particular embodiments, a method of the invention further comprises detecting in the sample at least one protein selected from the group consisting of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), THBS1 (SEQ ID NO:55), LMO7 (SEQ ID NO:56), TNC (SEQ ID NO:57), RBM39 (SEQ ID NO:58), ILVBL (SEQ ID NO:59), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64), ADAMDEC1 (SEQ ID NO:65), AMACR (SEQ ID NO:66), AMACR;C1QTNF3 (SEQ ID NO:67), ARID1A (SEQ ID NO:68), CEBPZ (SEQ ID NO:69), COL5A1 (SEQ ID NO:70), EFEMP2 (SEQ ID NO:71), FAM84B (SEQ ID NO:72), FKBP10 (SEQ ID NO:73), FKBP9 (SEQ ID NO:74), GPRC5A (SEQ ID NO:75), KPNA2 (SEQ ID NO:76), MMP1 (SEQ ID NO:77), PNMA5 (SEQ ID NO:78), POLR1C (SEQ ID NO:79), SPARC (SEQ ID NO:80), UBAP2 (SEQ ID NO:81), UCK2 (SEQ ID NO:82) and WDR74 (SEQ ID NO:83). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, a method of the invention further comprises use of at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), THBS1 (SEQ ID NO:55), LMO7 (SEQ ID NO:56), TNC (SEQ ID NO:57), RBM39 (SEQ ID NO:58), ILVBL (SEQ ID NO:59), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64), ADAMDEC1 (SEQ ID NO:65), AMACR (SEQ ID NO:66), AMACR; C1QTNF3 (SEQ ID NO:67), ARID (SEQ ID NO:68), CEBPZ (SEQ ID NO:69), COL5A1 (SEQ ID NO:70), EFEMP2 (SEQ ID NO:71), FAM84B (SEQ ID NO:72), FKBP10 (SEQ ID NO:73), FKBP9 (SEQ ID NO:74), GPRC5A (SEQ ID NO:75), KPNA2 (SEQ ID NO:76), MMP1 (SEQ ID NO:77), PNMA5 (SEQ ID NO:78), POLR1C (SEQ ID NO:79), SPARC (SEQ ID NO:80), UBAP2 (SEQ ID NO:81), UCK2 (SEQ ID NO:82) and WDR74 (SEQ ID NO:83). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the cancer is a cancer other than colorectal cancer and the at least one protein includes at least one of FAM62B (SEQ ID NO:51), SLC1A5 (SEQ ID NO:52), RSL1D1 (SEQ ID NO:53), LYZ (SEQ ID NO:54), TNC(SEQ ID NO:57), RBM39 (SEQ ID NO:58), ERO1L (SEQ ID NO:60), LOC442497 (SEQ ID NO:61), TCOF1 (SEQ ID NO:62), NAMPT (SEQ ID NO:2), SERPINB9 (SEQ ID NO:63), HSDL2 (SEQ ID NO:64).
In particular embodiments, the level of the at least one protein in the biological or test sample is increased by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, relative to the reference level. In particular embodiments, the reference level is representative of the level of the same protein in non-diseased tissue. In particular embodiments, the reference level is representative of the level of the same protein in a particular stage or form of cancer. In particular embodiments, the reference level is representative of the level of the same protein in a cancer having a known prognosis.
In another aspect, the invention provides a method of treating cancer, the method comprising administering to a subject in need thereof at least one pharmaceutical agent, wherein the pharmaceutical agent specifically interacts with at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20), CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49), CISD1 (SEQ ID NO:50), LYZ (SEQ ID NO:54), LOC442497; SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93). Each of the aforementioned agents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In a particular embodiment, the at least one protein is selected from the group consisting of KIAA0152 (SEQ ID NO:1), LYZ (SEQ ID NO:54), LOC442497; SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93). In particular embodiments, the pharmaceutical agent specifically interacts with KIAA0152 (SEQ ID NO:1). Each of the aforementioned reagents represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the pharmaceutical agent is selected from an antibody and an antibody mimetic. In particular embodiments, the pharmaceutical agent further comprises a cytotoxic moiety, such as a plant toxin, a bacterial toxin, a radioactive moiety or a chemotherapeutic agent. In particular embodiments, the pharmaceutical agent is selected from a chemical conjugate and fusion protein.
In another aspect, the invention provides an antigen composition comprising at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20), CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49), CISD1 (SEQ ID NO:50), LYZ (SEQ ID NO:54), LOC442497; SLC3A2 (SEQ ID NO:61), DMBT1 (SEQ ID NO:84), NUCB1 (SEQ ID NO:85), GGH (SEQ ID NO:86), AGR3 (SEQ ID NO:87), TM9SF2 (SEQ ID NO:88), SYK (SEQ ID NO:89), GCA (SEQ ID NO:90), HDLBP (SEQ ID NO:91), C1QBP (SEQ ID NO:92) and CLIC1 (SEQ ID NO:93), or an immunogenic fragment of said at least one protein. Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In a particular embodiment, the composition comprises at least one of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50), or an immunogenic fragment thereof. Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In a particular embodiment, the composition comprises KIAA0152 (SEQ ID NO:1) or an immunogenic fragment thereof. In a particular embodiment, a method of preventing or treating cancer in a subject in need thereof comprises administering to the subject an effective amount of said antigen composition. Further provided is an antigen composition as previously specified; for use in preventing or treating cancer.
In another aspect, the invention provides a kit for detecting, staging or prognosing cancer, the kit comprising at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19), CCT4 (SEQ ID NO:20); CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). Each of the aforementioned proteins represents a separate embodiment of the invention and may be used independently from or in combination with any of the others.
In particular embodiments, the kit is for detecting pre-cancerous polyps or early stage colorectal cancer and comprises at least one reagent suitable for detecting the level of at least one protein selected from the group consisting of CPT2 (SEQ ID NO:21), ARL1 (SEQ ID NO:22), PFKL (SEQ ID NO:23), GOT2 (SEQ ID NO:24), AP1G1 (SEQ ID NO:25), STRBP (SEQ ID NO:26), CLCA1 (SEQ ID NO:27), CYFIP1 (SEQ ID NO:28), COQ9 (SEQ ID NO:29), NDUFA9 (SEQ ID NO:30), ALDH7A1 (SEQ ID NO:31), HMGCS1 (SEQ ID NO:32), NNT (SEQ ID NO:33), PRDX5 (SEQ ID NO:34), PCCB (SEQ ID NO:35), COPZ1 (SEQ ID NO:36), BAX (SEQ ID NO:37), ACAD9 (SEQ ID NO:38), UBXD8 (SEQ ID NO:39), HMGCS2 (SEQ ID NO:40), SLC25A3 (SEQ ID NO:41), SLC25A11 (SEQ ID NO:42), PDCD6 (SEQ ID NO:43), UCRC (SEQ ID NO:44), DEFA6 (SEQ ID NO:45), DYNC1H1 (SEQ ID NO:46), HK1 (SEQ ID NO:47), CYFIP2 (SEQ ID NO:48), DCI (SEQ ID NO:49) and CISD1 (SEQ ID NO:50). In particular embodiments, the kit comprises a reagent suitable for detecting the level of KIAA0152 (SEQ ID NO:1).
According to some embodiments the present invention excludes proteins known to be associated with colorectal cancers. According to other embodiments the methods of the present invention exclude proteins that were known to be associated with other types of cancers.
Other objects, features and advantages of the present invention will become clear from the following description and examples.
The present invention provides methods of identification of cancerous cells by detection of levels of particular proteins, also referred to herein as “cancer-associated proteins or “molecular markers”, which have been found to be differentially expressed in cancer tissue, in particular, colorectal cancer tissue. Details of the subject cancer-associated proteins disclosed herein are provided in Tables 1 and 3-7. Some of the identified proteins for which significantly elevated levels were detected in colorectal cancer tissue as compared to normal colorectal tissue have not been previously disclosed to be associated with cancerous disease. Examples of such proteins are indicated in Table 1 as denoted by the symbol “+” in the column labeled “Newly identified as cancer-associated” and include the proteins FAM62B, SLC1A5. RSL1D1, LYZ, TNC, RBM39, ILVBL, ERO1L, LOC442497, TCOF1, NAMPT, SERPINB9 and HSDL2. Yet other proteins, in particular THBS1 and LMO7, have been disclosed in the prior art to be down-regulated in cancerous conditions, whereas the inventors of the present invention now disclose significantly increased levels of these proteins in cancer tissue, indicative of up-regulation. These proteins are indicated in Table 1 as denoted by the symbol “UR” in the column labeled “Newly identified as cancer-associated”. Yet other proteins were detected at significant levels in colorectal cancer tissue but were completely absent from healthy colorectal tissue in the same patients. Such proteins are indicated in Table 1 as denoted by the symbol “+” in the column labeled “Detected in cancerous but not healthy” and include the proteins AMACR, AMACR; C1 QTNF3, COL5A1, FKBP10, FKBP9, GPRC5A, POLR1c, SPARC and UBAP2.
Table 3 list proteins which are highly expressed in colorectal cancer, both in pre-cancerous polyps and in more advanced stages of the disease. Accordingly, any of the proteins listed in Table 3 represent candidates for use as diagnostic markers of colorectal cancer, which can be indicative of any of pre-cancerous lesions, early stage or late stage forms of the disease. Two currently preferred protein markers are KIAA0152 and NAMPT.
A quantitative analysis of some of the proteins listed in Table 3 is shown in
Table 4 lists proteins which appear to be highly expressed in pre-cancerous polyps, yet their expression tends to be diminished in more advanced stages of the disease. Accordingly, proteins listed in Table 4 represent promising candidates for use as very early diagnostic markers to identify individuals at risk of developing colorectal cancer.
Identification and quantification of a panel of tumor-associated proteins such as those listed in Tables 1 and 3-6, provides a specific means of detecting colorectal cancer in a subject, as well as means of prognosing and staging previously diagnosed colorectal cancers. Such methods can conveniently be carried out using detectably-labeled reagents which specifically bind the selected target proteins. Typically such reagents comprise monoclonal antibodies or small chemical mimetics thereof. In some cases however, it may be advantageous to employ nucleic acids complementary to mRNA species encoding the target proteins so as to quantitate expression activity which may be correlated with the corresponding protein levels. Furthermore, therapeutic pharmaceutical agents directed against tumor-associated proteins, for example, those disclosed in Table 7 herein, may be prepared, for example monoclonal antibodies conjugated to cytotoxic moieties, for use in targeted therapy of colorectal cancer.
TABLE 1
Detected
Newly
in
IPI
identified
cancerous
Acc.
as cancer-
but not
Protein Name
No.
associated
healthy
ABCF2 ATP-binding cassette, sub-
68506
family F, member 2 isoform b
ACIN1 Isoform 1 of Apoptotic
7334
chromatin condensation inducer in the
nucleus
ACOT7 Isoform 1 of Cytosolic acyl
10415
coenzyme A thioester hydrolase
ADAMDEC1 ADAM DEC1
4480
+
precursor
ADO 2-aminoethanethiol
45939
dioxygenase
ADSS Adenylosuccinate synthetase
26833
isozyme 2
AK3 GTP:AMP phosphotransferase
465256
mitochondrial
ALG5 Dolichyl-phosphate beta-
2506
glucosyltransferase
AMACR Alpha-methylacyl-CoA
847727
+
racemase
AMACR; C1QTNF3 alpha-
5918
+
methylacyl-CoA racemase isoform 1
ANXA3 Annexin A3
24095
ARHGEF1 Isoform 1 of Rho guanine
647786
nucleotide exchange factor 1
ARID1A Isoform 1 of AT-rich
643722
+
interactive domain-containing protein
1A
ARRDC1 Arrestin domain containing 1
514937
ATAD3A Isoform 2 of ATPase
295992
family AAA domain-containing
protein 3A
ATP6AP2 Renin receptor precursor
168884
ATP6V0A1 97 kDa protein
892784
ATP6V1E1 vacuolar H+ ATPase E1
719806
isoform b
BMS1 Ribosome biogenesis protein
6099
BMS1 homolog
BPI Bactericidal/permeability-
552280
increasing protein
BUD31 Protein BUD31 homolog
13180
BXDC1 Brix domain containing 1
644504
C14orf21 Pumilio domain-containing
216999
protein C14orf21
C1orf116 Isoform 1 of Specifically
28392
androgen-regulated gene protein
C1R; ACYP1; C17orf13 Complement
296165
C1r subcomponent precursor
C20orf43 UPF0549 protein C20orf43
297121
C2orf47 Uncharacterized protein
291751
C2orf47, mitochondrial precursor
C3orf64 Isoform 1 of
396231
Uncharacterized glycosyltransferase
AER61 precursor
C7orf24 Uncharacterized protein
31564
C7orf24
C8orf55 Uncharacterized protein
171421
C8orf55 precursor
CAD Putative uncharacterized protein
893035
CAD
CASP8 Uncharacterized protein
220725
CASP8
CBFB Core-binding factor subunit
16746
beta
CEACAM5 Carcinoembryonic
27486
antigen-related cell adhesion
molecule 5 precursor
CEBPZ CCAAT/enhancer-binding
306723
+
protein zeta
CHD4 Isoform 2 of Chromodomain-
455210
helicase-DNA-binding protein 4
CLPB Isoform 2 of Caseinolytic
216192
peptidase B protein homolog
COL12A1 Isoform 1 of Collagen
329573
alpha-1(XII) chain precursor
COL5A1 Collagen alpha-1(V) chain
844090
+
precursor
COMT Isoform Soluble of Catechol
375513
O-methyltransferase
CSTF1 Cleavage stimulation factor
11528
50 kDa subunit
CTSG Cathepsin G precursor
28064
DCK Deoxycytidine kinase
20454
DDX18 ATP-dependent RNA
301323
helicase DDX18
DEK 48 kDa protein
871695
DHCR7 7-dehydrocholesterol
294501
reductase
DHODH Dihydroorotate
24462
dehydrogenase, mitochondrial
precursor
DHX30 DEAH (Asp-Glu-Ala-His)
477295
box polypeptide 30 isoform 2
DIAPH1 Diaphanous homolog 1
884341
DMBT1 Isoform 1 of Deleted in
99110
malignant brain tumors 1 protein
precursor
DNAJA3 Isoform 2 of DnaJ homolog
179187
subfamily A member 3, mitochondrial
precursor
DNAJC19 10 kDa protein
795263
DPEP1 Dipeptidase 1 precursor
59476
EFEMP2 Mutant p53 binding protein
556657
+
1 variant (Fragment)
EI24 Isoform 2 of Etoposide-induced
23185
protein 2.4 homolog
EIF2B3 Isoform 1 of Translation
6504
initiation factor eIF-2B subunit
gamma
EIF2S2 Eukaryotic translation
21728
initiation factor 2 subunit 2
ERO1L ERO1-like protein alpha
386755
+
precursor
EXOC2 Exocyst complex component 2
783559
F11R Junctional adhesion molecule A
1754
precursor
FAM62B Isoform 2 of Extended
409635
+
synaptotagmin-2
FAM84B Protein FAM84B
64666
+
FAP Isoform 1 of Seprase
295461
FASN Fatty acid synthase
26781
FASTKD5 FAST kinase domain-
414973
containing protein 5
FDFT1 Squalene synthetase
20944
FERMT1 Isoform 1 of Fermitin
304754
family homolog 1
FKBP10 FK506-binding protein 10
303300
+
precursor
FKBP9 FK506-binding protein 9
182126
+
precursor
FOXK1 Isoform 1 of Forkhead box
556645
protein K1
FRG1 Protein FRG1
4655
FYB FYN binding protein (FYB-
73110
120/130) isoform 1
GCA Grancalcin
4524
GEMIN5 Gem-associated protein 5
291783
GGH Gamma-glutamyl hydrolase
23728
precursor
GLRX3 Glutaredoxin-3
8552
GLT25D1 Glycosyltransferase 25
168262
family member 1 precursor
GMPS GMP synthase
29079
GNL3 Isoform 1 of Guanine
306380
nucleotide-binding protein-like 3
GPR89B; GPR89A Isoform 1 of
472858
Protein GPR89
GPRC5A Retinoic acid-induced
22624
+
protein 3
GPX2 Glutathione peroxidase 2
298176
GTF2F1 General transcription factor
17450
IIF subunit 1
GTF2I Isoform 2 of General
293242
transcription factor II-I
HCFC1 Uncharacterized protein
641743
HCFC1
HM13 Isoform 1 of Minor
152441
histocompatibility antigen H13
HSDL2 Isoform 1 of Hydroxysteroid
414384
+
dehydrogenase-like protein 2
HSPH1 Isoform Beta of Heat shock
218993
Protein 105 kDa
ICAM1 Intercellular adhesion
8494
molecule 1 precursor
IGFBP7 Insulin-like growth factor-
16915
binding protein 7 precursor
IKIP IKIP2
401791
ILVBL Isoform 1 of Acetolactate
554541
synthase-like protein
IPO7 Importin-7
7402
ISLR Immunoglobulin superfamily
23648
containing leucine-rich repeat protein
precursor
KIAA0020 Pumilio domain-
791325
containing protein KIAA0020
KIAA0241 Isoform 1 of Protein
397348
KIAA0241
KIAA1219 Isoform 3 of Protein
410120
KIAA1219
KPNA2 Karyopherin alpha 2
789457
+
LACTB2 Beta-lactamase-like protein 2
6952
LCN2 Lipocalin 2
643623
LEPRE1 Isoform 3 of Prolyl 3-
45839
hydroxylase 1 precursor
LMO7 Isoform 3 of LIM domain only
291802
UR
protein 7
LOC442497; SLC3A2 solute carrier
554722
+
family 3 (activators of dibasic and
neutral amino acid transport), member
2 isoform e
LOC731605 similar to BCL2-
886854
associated transcription factor 1
isoform 2
LTBP2 Latent-transforming growth
292150
factor beta-binding protein 2
precursor
LTF Lactotransferrin precursor
848342
LYZ Lysozyme C precursor
19038
+
MAN1A2 Uncharacterized protein
743100
MAN1A2
MCM2 DNA replication licensing
184330
factor MCM2
MCM3 DNA replication licensing
13214
factor MCM3
MCM5 DNA replication licensing
18350
factor MCM5
MCM6 DNA replication licensing
31517
factor MCM6
MCM7 Isoform 1 of DNA replication
299904
licensing factor MCM7
MMP1 Interstitial collagenase
8561
+
precursor
MMP2 72 kDa type IV collagenase
27780
precursor
MMP9 Matrix metalloproteinase-9
27509
precursor
MOCS3 Molybdenum cofactor
4489
synthesis protein 3
MPO Isoform H7 of Myeloperoxidase
236556
precursor
MRPS6 Mitochondrial 28S ribosomal
305668
protein S6
MTA2 Metastasis-associated protein
171798
MTA2
MUC16 Mucin-16
103552
NAMPT Isoform 1 of Nicotinamide
18873
+
phosphoribosyltransferase
NAT10 N-acetyltransferase 10
300127
NCBP1 Nuclear cap-binding protein
19380
subunit 1
NCF4 Isoform 1 of Neutrophil
14338
cytosol factor 4
NEBL Nebulette variant 4
872370
NIP7 Isoform 1 of 60S ribosome
7175
subunit biogenesis protein NIP7
homolog
NOC2L Nucleolar complex protein 2
411886
homolog
NOC4L Nucleolar complex protein 4
31661
homolog
NOL10 Isoform 1 of Nucleolar
29513
protein 10
NQO1 NAD
12069
NUDCD1 Isoform 2 of NudC
306398
domain-containing protein 1
NUP188 Isoform 1 of Nucleoporin
477040
NUP188 homolog
NUP210 Isoform 1 of Nuclear pore
291755
membrane glycoprotein 210 precursor
NXF1 Nuclear RNA export factor 1
33153
OCIAD2 Isoform 1 of OCIA domain-
555902
containing protein 2
OLFM4 Olfactomedin-4 precursor
22255
OSBPL2 Isoform 1 of Oxysterol-
14137
binding protein-related protein 2
OTUD6B OTU domain containing
182180
6B
PAICS Multifunctional protein ADE2
217223
PARP14 poly (ADP-ribose)
291215
polymerase family, member 14
PCNA Proliferating cell nuclear
21700
antigen
PEX14 Peroxisomal membrane
25346
protein PEX14
PHF6 Isoform 1 of PHD finger
395568
protein 6
PKP3 Plakophilin-3
26952
PLOD3 Procollagen-lysine, 2-
30255
oxoglutarate 5-dioxygenase 3
precursor
PNMA5 Paraneoplastic antigen-like
514588
+
protein 5
POLR1C Isoform 1 of DNA-directed
5179
+
RNA polymerases I and III subunit
RPAC1
PROM1 Prominin-1 precursor
12540
PSAT1 Isoform 1 of Phosphoserine
1734
aminotransferase
PTDSS1 Phosphatidylserine synthase 1
10746
PTK7 Tyrosine-protein kinase-like 7
298292
precursor
PUF60 Isoform 5 of Poly
856076
PYCR1 pyrroline-5-carboxylate
376503
reductase 1 isoform 2
PYCR2 Pyrroline-5-carboxylate
470610
reductase 2
RAD21 Double-strand-break repair
6715
protein rad21 homolog
RANBP3 Isoform 2 of Ran-binding
456728
protein 3
RBM12 RNA-binding protein 12
550308
RBM39 Isoform 2 of RNA-binding
215801
+
protein 39
RCC1 regulator of chromosome
787306
condensation 1 isoform b
RCC2 Protein RCC2
465044
RCN1 Reticulocalbin-1 precursor
15842
RDBP Negative elongation factor E
858
REEP6 Receptor expression-
647161
enhancing protein 6
REG1A Lithostathine-1-alpha
9027
precursor
REG1B Lithostathine-1-beta
9197
precursor
RFC2 Isoform 2 of Replication factor
218280
C subunit 2
RFC5 Replication factor C subunit 5
31514
RRM1 Ribonucleoside-diphosphate
13871
reductase large subunit
RRP1 RRP1-like protein
550766
RRS1 Ribosome biogenesis
14253
regulatory protein homolog
RSL1D1 RSL1D1 protein
642046
+
S100A11 Protein S100-A11
13895
S100A12 Protein S100-A12
218131
S100A8 Protein S100-A8
7047
S100A9 Protein S100-A9
27462
SAE1 SUMO-activating enzyme
33130
subunit 1
SDCCAG10 Isoform 1 of Peptidyl-
25174
prolyl cis-trans isomerase
SDCCAG10
SERPINB5 Serpin B5 precursor
783625
SERPINB9 Serpin B9
32139
+
SERPINH1 Serpin H1 precursor
32140
SET Isoform 2 of Protein SET
301311
SLC1A5 Neutral amino acid
19472
+
transporter B
SLC25A15 Mitochondrial ornithine
3389
transporter 1
SLC2A1 Solute carrier family 2,
220194
facilitated glucose transporter
member 1
SORD 11 kDa protein
791243
SPARC SPARC precursor
14572
+
SQSTM1 Isoform 1 of
179473
Sequestosome-1
SRM Spermidine synthase
292020
SRRM2 Isoform 1 of Serine/arginine
782992
repetitive matrix protein 2
SSBP1 Single-stranded DNA-binding
29744
protein, mitochondrial precursor
SYK Isoform Long of Tyrosine-
18597
protein kinase SYK
TBC1D2B Isoform 1 of TBC1
550733
domain family member 2B
TCOF1 Isoform 2 of Treacle protein
298696
+
TFRC Transferrin receptor protein 1
22462
TH1L Isoform NELF-D of Negative
759539
elongation factor C/D
THBS1 Thrombospondin-1 precursor
296099
UR
THBS2 Thrombospondin-2 precursor
18769
THOC6 Isoform 1 of THO complex
328985
subunit 6 homolog
TIMP1 TIMP metallopeptidase
642739
inhibitor 1
TJP2 Isoform A1 of Tight junction
3843
protein ZO-2
TM9SF4 Isoform 2 of
885106
Transmembrane 9 superfamily
member 4 precursor
TNC Isoform 1 of Tenascin precursor
31008
+
TOMM34 Mitochondrial import
9946
receptor subunit TOM34
TOP1 DNA topoisomerase 1
413611
TOP2A Isoform 2 of DNA
414101
topoisomerase 2-alpha
TPR nuclear pore complex-associated
742682
protein TPR
TRMT6 Isoform 1 of tRNA
99311
UBAP2 Ubiquitin-associated protein 2
171127
+
UBE2O Ubiquitin-conjugating
783378
enzyme E2 O
UCK2 Isoform 1 of Uridine-cytidine
65671
+
kinase 2
UQCC 34 kDa protein
872061
URB1 Nucleolar pre-ribosomal-
297241
associated protein 1
UTP20 Small subunit processome
4970
component 20 homolog
VAMP7 Isoform 3 of Vesicle-
401804
associated membrane protein 7
WDR43 WD repeat-containing
55954
protein 43
WDR74 Isoform 1 of WD repeat-
18192
+
containing protein 74
XPO5 Isoform 1 of Exportin-5
640703
XPOT Exportin-T
306290
YLPM1 YLP motif containing 1
165434
The terms “subject” and “patient” as used herein refer to any single subject for which cancer detection, prognosis, staging or therapy is desired, including humans and non-human mammals, such as primate, bovine, ovine, canine, feline and rodent mammals. Also included are subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.
The terms “cancer detection” and “cancer diagnosis” and related grammatical terms, such as “detecting cancer” and “diagnosing cancer”, respectively, are used herein interchangeably to refer to any of determination of a subject's susceptibility to a malignant cancer disease; determination as to whether a subject is presently affected by a malignant cancer disease; determination of a subject's stage of cancer, determination of and monitoring the effect on the cancer in response to anti-cancer therapy.
The term “characteristics of a cancerous or pre-cancerous growth” as used herein refers to one or more molecular, physiological, histological, clinical or other properties which may be used to define the nature and behavior of the growth.
The terms “cancer”, “neoplasm”, “tumor”, “growth” and the like are used interchangeably herein to refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In general, cells of interest for detection or treatment in the present application include pre-malignant (e.g., benign hyperplasic), malignant, metastatic, and non-metastatic cells.
The term “prognosis” as used herein refers to the expected or predicted outcome of a disease, such as a cancer, in a patient following diagnosis. A prognosis may predict the relative chance of disease progression, arrest or cure. A prognosis may be established on the basis of prognostic indicators specific for a particular disease. Prognostic indicators in cancer may include for example, the grade and stage of cancer at initial diagnosis, the genetic make-up of the patient, the presence and level of cancer-associated antigens in the tumor, and patient responsiveness to a particular therapy.
The terms “biological sample” and “test sample” as used herein encompass a variety of types of biological materials that can be used in the methods of the invention. The sample may be procured from the body of an individual or investigated without removal from the body of an individual. The term encompasses solid tissue samples, such as from biopsy specimens, tumors or tumor metastases, or tissue cultures or cells derived there from and the progeny thereof. The terms also encompass blood and other liquid samples of biological origin, such as nipple aspirate fluid, lymph node aspirate, mucosal fluid, cervical wash, lacrimal duct fluid, urine, saliva, pleural effusion and sputum. The terms encompass samples that have been manipulated in any way after their procurement, such as by lysis, treatment with reagents, solubilization, or enrichment for certain components. Also included are clinical samples, cells in cell culture, cell supernatants and cell lysates. It is to be explicitly understood that in accordance with the invention, a biological or test sample may be obtained i.e. removed, from the body of a subject, or accessed in vivo, for example by contacting with a specific reagent or apparatus.
As used herein, a biological or test sample that is “obtained from a subject”, means that the sample is removed from the body of the subject, and any subsequent analysis thereof may be performed outside the body for example under in vitro or ex vivo conditions. When however, a biological or test sample is “assessed in vivo” or “accessed in vivo” it means that the sample is maintained within the body of the subject and direct or indirect contact is established using any suitable means, such as via a reagent, composition, device or apparatus. Subsequent analysis of the accessed sample is performed under in vivo conditions, which can include conditions of local or general anaesthesia. Means of accessing and assessing a sample in vivo include for example, contacting the tissue or organ or region thereof of interest with a pharmaceutical composition, a swallowable endoscopy capsule or an endoscopy probe.
The term “a normal biological sample of the same type” as used herein refers to a non-diseased sample consisting of the same biological material or type of tissue and/or cells e.g. blood, colorectal tissue, as that of the test sample. The normal biological sample may be that from a single individual, including the subject in which cancer detection, prognosing or characterizing is performed, or from a group of individuals of known healthy status. Accordingly, the level of a protein in a normal biological sample may be obtained from a single determination or may advantageously represent a statistical average of multiple determinations, such as from a group of healthy individuals or from multiple healthy tissue sites in a single individual.
The term “a control sample” as used herein refers to the standard provided by either a normal i.e. non-diseased sample or group of samples, or a sample or group of samples corresponding to an established form, type, stage or grade of a disease, in particular a cancer disease. Accordingly, the level of a protein in a control sample may be obtained from a single determination or may advantageously represent a statistical average of multiple determinations, for example from a group of healthy individuals, or from a group of diseased individuals established to have the same form, type, stage or grade of a cancer disease.
The term “non-diseased tissue” as used herein refers to tissue which is determined to be free of a cancer disease, on the basis of any technique known in the art, for example, histological and immuno-histochemical investigation. It is to be understood that diseased and non-diseased tissue of the same tissue type may be present in close proximity in a individual patient.
The term “immunogenic fragment” as used herein in reference to a protein refers to a portion of the protein which is capable of inducing an immune response, such as antibody production, following administration to an individual.
As used herein, the terms “a protein associated with cancer”, “tumor associated protein”, “molecular marker” and the like, interchangeably refer to a protein that is present at relatively higher or lower levels in a cancer cell relative to a normal cell of the same type (e.g., as in protein associated with colon cancer).
As used herein the terms “nucleic acid encoding a protein,” encompass polynucleotides and polypeptides respectively having sequence similarity or sequence identity to the respective gene and gene products having the accession numbers of the particular protein referred to, of at least about 65%, preferably at least about 80%, more preferably at least about 85%, and can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Sequence similarity and sequence identity are calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. In general, percent sequence identity is calculated by counting the number of residue matches (e.g., nucleotide residue or amino acid residue) between the query and test sequence and dividing total number of matches by the number of residues of the individual sequences found in the region of strongest alignment, as is known in the art. Algorithms for computer-based sequence analysis are known in the art, such as BLAST (see, e.g., Altschul et al., J. Mol. Biol., 215:403-10 (1990)), particularly the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular).
The terms “protein” and “polypeptide” are used interchangeably herein to refer to polymeric forms of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
As used herein, a “fusion protein” or “chimeric peptide” refers to a protein or polypeptide which comprises at least a portion of a first naturally occurring protein or polypeptide fused to least a portion of a second protein or polypeptide. For example, a fusion protein for targeting FAM62B may include a portion or all of an anti-FAM62B antibody fused with another peptide or polypeptide such as a protein label moiety or a cytotoxic protein.
The term “antibody” as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies, humanized antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies and antibody fragments (e.g., F(ab′)2, Fab′, Fab, Fv) so long as they bind specifically to a target antigen or epitope of interest.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries, as is known in the art, for example using techniques such as those described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).
Furthermore, monoclonal antibodies specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see for example U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
The term “reagent suitable for detecting the level of a protein” as used herein refers to a reagent which either specifically binds the protein itself or is complementary to a nucleic acid molecule that is involved in expression of the protein. Particularly suitable examples of reagents which specifically bind to proteins are antibodies and chemical mimetics thereof having similar level of binding affinity and/or avidity. Nucleic acid reagents which are complementary to mRNA encoding a protein of interest are suitable for determining the level of the protein, typically by correlating the amount of mRNA detected with the corresponding level of protein product produced in a suitable translation system.
The terms “specifically interacts” and “specifically binds” are used herein interchangeably to refer to high avidity and/or high affinity binding of a reagent, such as an antibody to a specific polypeptide or epitope thereof, such as for example, an epitope of any of the proteins FAM62B, SLC1A5, RSL1D1, LYZ, RBM39 and TCOF1. Antibody binding to its epitope is stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest. Antibodies which bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g., by use of appropriate controls.
The term “primary antibody” as used herein refers to an antibody which binds specifically to the target protein antigen in a biological or test sample. A primary antibody is generally the first antibody used in an immunoassay procedure. In some embodiments, the primary antibody is the only antibody used in an immunoassay procedure.
The term “secondary antibody” as used herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent, if any. The secondary antibody is typically directed against the Fc portion of the immunoglobulin type of the primary antibody (e.g., anti-mouse Fc antibody).
The terms “polynucleotide” and “nucleic acid” are used interchangeably herein to refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides, including but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Further include are mRNA or cDNA that comprise intronic sequences (see, e.g., Niwa et al. (1999) Cell 99(7):691-702). The backbone of the polynucleotide can comprise sugars and phosphate groups (as typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component, capping, substitution of one or more of naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
The term “complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules or a DNA/RNA hybrid. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).
The term “hybridization” as used herein encompasses any process by which a strand of nucleic acid joins with a complementary strand through base pairing (see for example, Coombs, Dictionary of Biotechnology, Stockton Press, New York N.Y. (1994)). Accordingly, a hybridization assay is a quantitative means of determining the extent of hybridization between a nucleic acid in a test sample, such as a tissue sample, and a nucleic acid probe corresponding to at least a fragment of a gene (DNA or RNA) encoding a protein of interest, such as a cancer associated protein. “Stringency” typically occurs in a range from about Tm −5° C. (5° C. below the Tm of the nucleic acid probe) to about 20° C. to 25° C. below Tm. As will be understood by those of skill in the art, a stringency hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
Amplification as carried out in the polymerase chain reaction technologies is described in Dieffenbach et al., PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y. (1995), and may be performed prior to or as part of a hybridization assay.
As used herein, the term “differentially expressed” generally refers to a polynucleotide and/or the corresponding protein that is expressed at levels in a test cell that differ significantly from levels in a reference or control cell, e.g., a cancer-associated protein as disclosed herein is found at levels at least about 50% to about 100% increased, generally at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold or more increased in a cancerous cell when compared with a cell of the same type that is not cancerous. The comparison can be made between two tissues, for example, if one is using in situ hybridization or another assay method that allows some degree of discrimination among cell types in the tissue. The comparison may also be made between cells removed from their tissue source, or cells maintained in their native state in vivo. “Differential expression” refers to both quantitative, as well as qualitative, differences in the genes' temporal and/or cellular expression patterns among, for example, normal and neoplastic tumor cells, and/or among tumor cells which have undergone different tumor progression events.
The terms “correspond to” or “represents” as used in, for example, the phrase “polynucleotide corresponds to a differentially expressed gene” are used to refer to the relationship between a given polynucleotide and the gene from which the polynucleotide sequence is derived (e.g., a polynucleotide that is derived from a coding region of the gene, a splice variant of the gene, an exon, and the like) or to which the polynucleotide hybridizes to under stringent conditions.
The term “label” as used herein refers to a compound or composition which is conjugated or fused directly or indirectly to a reagent such as an antibody, a nucleic acid probe or a chemical agent and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
The term “mimetic” as used herein refers to any entity, including natural and synthesized inorganic or organic molecules, including recombinant molecules, that mimic the properties of the molecule of which it is a mimetic. Accordingly, a mimetic of a particular antibody has the same, similar or enhanced epitope binding properties of that antibody.
The term “gene” as used herein refers to any nucleic acid sequence or portion thereof with a functional role in encoding or transcribing a protein or regulating other gene expression. The gene may consist of all the nucleic acids responsible for encoding a functional protein or only a portion of the nucleic acids responsible for encoding or expressing a protein. The nucleic acid sequence may contain a genetic abnormality within exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences or unique adjacent regions to the gene.
The singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the reagent” includes reference to one or more reagents and equivalents thereof known to those skilled in the art, and so forth.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Detection, Prognosis and Management of Colorectal Cancer
The present invention is based on the discovery that specific proteins, including for example, KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D and CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, FAM62B, S100A8, S100A9, MPO, MCM2, LTF, OLFM4, FERMT1, CEACAM5, SLC1A5, THBS1, NAT10, RSL1D, LMO7, LYZ and MCM3 are present at significantly higher levels in cancerous or pre-cancerous colorectal tissue as compared to normal tissue of the same cell type, even in the same individual.
This discovery serves as the basis for identification of cancerous tissue, as well as for staging and prognosing tumors, and development of therapeutic agents which target the particular cancer-associated markers. Detection of the markers disclosed herein and/or the genes encoding them, enables early diagnosis based on molecular changes leading to carcinogenesis and/or decision making in disease management. For example, a relatively increased level of a particular protein, such as FAM62B, compared to normal cells or tissues of the same type can be indicative of a poorer prognosis, and therefore warrant more aggressive therapy (e.g., chemotherapy following surgery). The correlation of tumor specific markers with response to treatment and outcome in patients can define prognostic indicators that allow the design of tailored therapy based on the molecular profile of the tumor. These therapies include antibody targeting, antagonists (e.g., small molecules), and gene therapy. Determining colon cancer-specific protein levels and comparison of a patient's profile with known levels in normal tissue and variants of the disease allows a determination of optimal treatment strategies. The marker expression pattern can also be used to better classify (i.e. stage and grade), and thus diagnose and treat different forms of cancer. Furthermore, a protein identified as being differentially or specifically expressed in, one type of cancer may also have implications for development or risk of development of other types of cancer.
Colorectal cancer is one of the most common neoplasms in humans and perhaps the most frequent form of hereditary neoplasia. Prevention and early detection are key factors in controlling and curing colorectal cancer. Colorectal cancer begins as polyps, which are small, benign growths of cells that form on the inner lining of the colon. Over a period of several years, some of these polyps accumulate additional mutations and become cancerous. Multiple familial colorectal cancer disorders have been identified, as follows: 1) Familial adenomatous polyposis (FAP); 2) Gardner's syndrome; 3) Hereditary nonpolyposis colon cancer (HNPCC); and 4) Familial colorectal cancer in Ashkenazi Jews.
Staging is a process used in the medical arts to describe how advanced the cancerous state is in a patient. While staging systems vary with the types of cancer, they generally involve the “TNM” system: “T” indicates the type of tumor, “N” indicates whether the cancer has metastasized to nearby lymph nodes; and “M” indicates whether the cancer has metastasized to other parts of the body. Generally, if a cancer is only detectable in the area of the primary lesion without having spread to any lymph nodes it is called Stage I. If it has spread only to the closest lymph nodes, it is called Stage II. In Stage III, the cancer has generally spread to the lymph nodes in near proximity to the site of the primary lesion. Cancers that have spread to a distant part of the body, such as the liver, bone, brain or other site, are Stage IV, the most advanced stage.
The grade of a cancer describes how closely a tumor resembles normal tissue of its same type. The microscopic appearance of a tumor is used to identify tumor grade based on parameters such as cell morphology, cellular organization, and other markers of differentiation. As a general rule, the grade of a tumor corresponds to its rate of growth or aggressiveness, with undifferentiated or high-grade tumors generally being more aggressive than well differentiated or low-grade tumors. The following guidelines are generally used for grading tumors: GX, Grade cannot be assessed; G1, Well differentiated; G2, Moderately well differentiated; G3, Poorly differentiated; G4, Undifferentiated.
Methods of detection, prognosis, and characterization as disclosed herein are based on levels of at least one, and preferably a panel or multiplicity of cancer-associated proteins, in comparison to levels of the same protein(s) in suitable non-cancerous or cancerous control samples. For example, a detection of cancer may be enabled by the detection of a level of one or more proteins of interest in a sample, such as FAM62b, that is increased at least by 50% or 100% or greater or, alternatively by 2-fold, 3-fold 5-fold, 10-fold, 30-fold, or greater, relative to a normal non-cancerous sample of the same tissue type. The normal non-cancerous sample may be from the same individual as the test sample or from one or more different individuals. Preferably, the level in the normal non-cancerous sample is a statistical average of multiple determinations, such as from a group of healthy individuals or from multiple healthy tissue sites in a single individual.
Similarly, prognosis and characterization of the cancer may be established on the levels of proteins of interest relative to reference standards established for particular grades, stages and forms of cancer. For example, detection of a level of a particular protein which is 1.5-fold compared to that of the control may be taken to indicate a relatively positive prognosis and/or relatively non-aggressive type of cancer, whereas detection of a level of the same protein of 10-fold or more compared to that of the control may be taken to indicate a poorer prognosis and/or a substantially aggressive type of cancer.
Assay Systems and Methods
The methods of the invention may be carried out using various assay systems and methods for detection of the level of the protein of interest in a test biological sample. Suitable systems include those employing an immunoassay, a nucleic acid hybridization assay, a binding assay, an array, a phage display library, or a combination thereof.
Immunoassays
Immunoassays for detecting levels of specific binding between an antibody and its target antigen are known in the art and include for example, radioimmunoassay, (RIA), fluorescent immunoassay, (FIA) enzyme-linked immunosorbant assay (ELISA), immunohistochemistry (IHC) and fluorescent activated cell sorting (FACS) (see, e.g., Harlow and Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1999)). In general, an immunoassay may be either direct or indirect. In a direct assay, the binding of antibody to the target antigen is determined directly using a labeled reagent, such as a fluorescent labeled or an enzyme-labeled primary antibody, which can be detected without further antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody. Alternately, both the primary and secondary antibodies may be unlabeled and labeled tertiary antibody is employed. Where the antibody (primary, secondary or tertiary) is conjugated to an enzymatic label, a chromagenic or fluorogenic substrate is added to provide detection of the antigen.
The primary antibody used for detection of the protein(s) of interest is stably associated with e.g., directly or indirectly bound to, the surface of a solid support, e.g. column, microtiter plate, beads, membrane, typically made of glass, plastic, polysaccharides, nylon or nitrocellulose. A multiplicity of antibody specificities for detecting a panel of tumor-associated proteins may be simultaneously bound to the same support, such as in an array.
The test sample is allowed to contact the support during a period of incubation, generally following blocking of non-specific binding sites with non-interfering proteins such as bovine serum albumin. After incubation with each reagent e.g. blocking agent, primary antibody, secondary antibody, the support is washed to remove non-bound components. Determination of suitable reagents, conditions for washing, incubation etc. is within the ability of one of average skill in the art.
Immunoassays can also be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of tumor associated antigen. In situ detection can be accomplished by removing a histological sample from a subject, and contacting the sample with a labeled antibody. The antibody is typically contacted with the sample by overlaying the labeled antibody onto the sample. Through the use of such a procedure, the presence of the tumor associated antigen can be determined and/or the distribution of the antigen in the histological sample can be examined. Those of ordinary skill in the art will readily appreciate that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Detectable labels suitable for conjugation to antibodies and other binding reagents include radioisotopes, fluorescent labels, enzyme-substrate labels, chromogenic labels, chemiluminescent labels and colloidal gold particles.
Radioisotopes include for example, 35S, 14C, 125I, 32P and 131I. Fluorescent labels include for example, fluorescent molecules fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin (PE), phycocyanin, allophycocyanin, ortho-phthaldehyde, fluorescamine, peridinin-chlorophyll a (PerCP), Cy3 (indocarbocyanine), Cy5 (indodicarbocyanine), lanthanide phosphors, and the like.
Enzymatic labels include luciferases (e.g. firefly luciferase and bacterial luciferase), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of enzyme-substrate combinations include, for example: horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidine hydrochloride (TMB)); alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and β-D-galactosidase β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase).
A label may be indirectly conjugated with an antibody or other reagent, as is known in the art. For example, an antibody can be conjugated with biotin and any of the types of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin, and thus, the label can be conjugated with the antibody in an indirect manner. In some cases, detectable labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
Detection of bound, labeled antibody can be carried out by standard colorimetric, radioactive, photometric and/or fluorescent detection means. For fluorescent labels, signal can be detected by, for example, a scanning confocal microscope in photon counting mode. Appropriate scanning devices are described by, for example, U.S. Pat. Nos. 5,578,832 and 5,631,734. For antibodies labeled with biotin, the reaction can be treated with the appropriate streptavidin-conjugate (e.g., streptavidin-horseradish peroxidase, streptavidin-alkaline phosphatase, streptavidin-luciferase, and the like) and then treated with the appropriate reagents for calorimetric or photometric detection. For radiolabeled antibody, signal can be detected using a scintillation counter, phosphoimager or similar device.
Arrays
Cancer associated proteins in a sample may be detected using an array-based binding assay system. Such an array-based system may incorporate an immunoassay as described above, or incorporate small molecule chemical entities which specifically interact with particular cancer associated proteins. In either case, the solid substrate used for the array comprises a plurality of binding reagents attached to the substrate, wherein each binding reagent has specificity for a different cancer associated protein. The protein panel or “set” for which the array is predetermined to specifically bind and detect is characteristic of a particular disease, or stage of form of a disease. The binding reagents, are immobilized onto the substrate surface, preferably in a spatially addressable manner. The binding reagents may be antibodies, antibody fragments or small molecule chemical entities.
The nature and geometry of the solid substrate will depend upon a variety of factors, including, among others, the type of array (e.g., one-dimensional, two-dimensional or three-dimensional). Generally, the surface can be composed of any material which will permit immobilization of the binding reagents and which will not substantially degrade under the conditions used in the applications of the array.
The solid substrate used for the array may be in the form of beads, particles or sheets, and may be permeable or impermeable, depending on the type of array, wherein the surface is coated with a suitable material enabling binding of the binding reagents at high affinity. For example, for linear or three-dimensional arrays the surface may be in the form of beads or particles, fibers (such as glass wool or other glass or plastic fibers) or glass or plastic capillary tubes. For two-dimensional arrays, the solid surface may be in the form of plastic, micromachined chips, membranes, slides, plates or sheets in which at least one surface is substantially flat, wherein these surfaces may comprise glass, plastic, silicon, low cross-linked and high cross-linked polystyrene, silica gel, polyamide, and the like.
Fluorescence tagged beads are also an addressable (liquid) array in which each bead is tagged with a different set of fluorescent colors and bound with an antibody; specific to an array is detected with devices such as fluorescence scanners for arrays or FACS for beads.
The arrays used for the present invention may be of any desired size. The upper and lower limits on the size of the array are determined solely by the practical considerations of resolution, size of molecules expressed at each address and the like.
Either a population of discrete proteins is employed to form the array, such that each address presents a different molecule, or a single or a few addresses are employed with a similar protein. In many applications, redundancies in the spots are desirable for the purposes of acting as internal controls.
Technologies for the deposition of droplets containing protein binding reagents onto a suitable solid surface are known in the art. An ink-jet printing technology for deposition of small droplets while avoiding overlap or splatter is disclosed in U.S. Pat. No. 5,449,754.
In order to conduct array-based binding assays, the test sample is allowed to contact the array comprising a coated surface containing the anchored binding reagents. Following contact, the array is optionally washed, typically under conditions such that any complexes formed will remain immobilized on the solid surface and unbound material will be removed.
The detection of complexes anchored on the solid surface can be accomplished in a number of ways. In some embodiments, the non-immobilized sample is pre-labeled, and the detection is directed to label immobilized on the surface indicating that complexes were formed. In other embodiments, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non-immobilized sample (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). In another embodiment, the immobilized molecules of the microarray are labeled, the array can be scanned or otherwise analyzed for detectable assay signal, and the signal from each labeled spot, or alternatively from all spots, quantified.
An important consideration is the presence of an amount of a label at each position within the array that is proportional to the amount of molecule immobilized at that particular spot. Thus, it is important that the efficiencies of the coupling reactions which are used to immobilize the labeled molecules are substantially similar.
Virtually any label that produces a detectable, quantifiable signal and that is capable of being attached to an immobilized binding reagent on a substrate can be used in conjunction with the array of the invention. Suitable labels include: radioisotopes, fluorophores, chromophores, chemiluminescent moieties, as described above.
Preferably, the position of the label will not interfere with interaction between a desired sample and the immobilized molecules and with the detection in case of an interaction between the desired sample and an immobilized molecule of the array. Suitable methods of making labeled molecules are well known in the art.
In the case where each spot in the array contains an amount of a label or “tracer” proportional to the amount of molecules immobilized at the particular spot, the signals obtained from the arrays of the invention can be normalized. As a consequence, signal intensities from spots within a single array, or across multiple arrays, can be directly compared. A normalized signal of a particular spot may be defined by (It−Io)/Io, where It is the intensity of the signal of the spot after contacting with a sample of interest and Io is the intensity of the background signal of the spot before contacting with a sample of interest.
Various methods and devices for detection and analysis of the array are known in the art. Practically, any imaging system that is capable of detecting with a resolution appropriate to the size of the array features can be utilized. For example, a method for screening an array of proteins for interactions with a fluid sample is disclosed in U.S. Pat. No. 6,475,809. Imaging apparatus may be selected, for example, from ScanArray 4000 (General Scanning), Biochip Imager (Hewlett Packard), GMS 418 Array Scanner (Genetic Microsystems), GeneTAC 1000 (Genomic Solutions), Chip Reader (Virtek). Phosphorimager systems are available for detecting radiolabels, e.g. Cyclone (Packard Instrument Co.) and BAS-5000 (Fujifilm).
Hybridization Assays
Hybridization assays generally comprise contacting a sample containing nucleic acids (target nucleic acids) with a nucleic acid probe capable of hybridizing to tumor associated antigen nucleic acids, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
Suitable hybridization assays include, for example, Northern blots, dot blots, RT-PCR, and quantitative PCR. Such procedures can be performed in situ directly for example, in tissue sections (e.g., fixed and/or frozen) of subject tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Tumor associated antigen nucleic acids can be used as probes and/or primers for such procedures (see e.g., Nuovo, PCR In Situ Hybridization: Protocols and Applications, Raven Press, NY (1992)).
Detection of tumor associated antigen nucleic acids typically involves contacting and incubating nucleic acids from a test sample with one or more labeled nucleic acids, (i.e. “probes”) under conditions favorable for the specific annealing of the nucleic acids to their complementary sequences. Typically, the lengths of the nucleic acid reagents are at least 15 to 30 nucleotides. After incubation, all non-annealed nucleic acids are removed. The presence of bound i.e. hybridized, nucleic acids from the sample, if any such molecules exist, is then detected. Using such a detection scheme, the nucleic acid from the tissue or cell type of interest can be immobilized, to a solid support such as a membrane, or a plastic surface such as that on a microtiter plate or polystyrene beads.
Nucleic acid arrays can be used to monitor the expression of tumor associated genes, such as, for example, those corresponding to KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D and CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, FAM62B, S100A8, S100A9, MPO, MCM2, LTF, OLFM4, FERMT1, CEACAM5, SLC1A5, THBS1, NAT10, RSL1D, LMO7, LYZ and/or MCM3. For detection of a multiplicity of genes encoding distinct cancer associated proteins, an array of polynucleotide probes may be contacted with a sample of target nucleic acids to produce a hybridization pattern. The binding of the target nucleic acids to one or more probes of the array is then detected to obtain a qualitative and/or quantitative profile of expression of the tumor associated antigen gene.
A variety of different arrays can be used and are known in the art. The polymeric or probe molecules of the arrays can be polynucleotides or hybridizing derivatives or analogs thereof, including: nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phosphorothioate, methylimino, methyl-phosphonate, phosphoramidate, guanidine, and the like; nucleic acids in which the ribose subunit has been substituted, for example, hexose phosphodiester; peptide nucleic acids; and the like. The length of the probes will generally range from about 10 to about 1000 nucleotides, typically, from about 15 to about 150 nucleotides in length, but also possibly from about 150 to about 1000 nucleotides in length. The probes can be single or double stranded, usually single stranded, and can be PCR fragments amplified from cDNA. The probe molecules on the surface of the substrates will typically correspond to at least one of the tumor associated antigen genes and be positioned on the array at known locations so that positive hybridization events can be correlated to expression of a particular gene in the physiological source from which the target nucleic acid sample is derived. Because of the manner in which the target nucleic acid sample is generated, the arrays of probes will generally have sequences that are complementary to the non-template strands of the gene to which they correspond.
The substrate for the array can be fabricated from a variety of materials, including plastics, ceramics, metals, gels, membranes, glasses, and the like. The arrays can be produced according to any convenient methodology, such as pre-forming the probes and then stably associating them with the surface of the support or growing the probes directly on the support. A number of different array configurations and methods for their production are known to those of skill in the art and disclosed in, for example, U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637.
The target nucleic acid is typically contacted with the array under conditions sufficient for hybridization of target nucleic acid to probe to occur. Suitable hybridization conditions are well known to those of skill in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor, Cold Spring Harbor, N.Y. (2001)).
The amount of tumor associated antigen nucleic acids in the sample can be quantitated (see, e.g., U.S. Pat. No. 6,004,755). For example, the target nucleic acids in the sample can be end-labeled in a manner such that each of the target nucleic acids in the sample produces a signal of the same specific activity. By generating the same specific activity is meant that each individual target polynucleotide in the sample being assayed is labeled in a manner such that the molecule is capable of providing the same signal (e.g., the same intensity of signal) as every other labeled target in the sample. Each of the target nucleic acids generates a signal of the same specific activity because the number of labeled nucleotide bases in each of the target molecules is either identical or substantially the same.
The label is capable of providing a detectable signal, either directly or through interaction with one or more additional members of a signal producing system. Suitable detectable labels include radioactive, fluorescent and enzymatic labels as described above.
In some applications, it is desired to analyze populations of target nucleic acids from two or more samples. Such samples can be differentially labeled. Alternatively, target nucleic acids from different samples are separately contacted to identical probe arrays under conditions of hybridization, typically stringent hybridization conditions, such that labeled nucleic acids hybridize to their complementary probes on the substrate surface, and the target nucleic acids bound to the array separately detected. A set of standard nucleic acid molecules can optionally be used. For example, the standard nucleic acids can be provided by reverse transcribing standard RNA.
Following hybridization, a washing step is usually employed to remove non-specifically bound nucleic acid from the support surface, generating a pattern of hybridized nucleic acid on the substrate surface. Various wash solutions and protocols for their use are known to those of skill in the art.
Where the label on the target nucleic acid is not directly detectable, the array can be contacted with the other member(s) of the signal producing system that is being employed. For example, where the label on the target is biotin, the array can be contacted with streptavidin-fluorophore conjugate under conditions sufficient for binding between the specific binding member pairs to occur. Following contact, any unbound members of the signal producing system will then be removed (e.g., by washing).
The resultant hybridization pattern(s) of target nucleic acids bound to the array can be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid. For example, detection means can include scintillation counting, autoradiography, fluorescence measurement, colorimetric measurement, light emission measurement, and the like.
Prior to detection or visualization, the array of hybridized target/probe complexes can be optionally treated with an endonuclease, for example, mung bean nuclease, S1 nuclease, and the like. The endonuclease degrades single stranded, but not double stranded DNA.
Following detection or visualization, the hybridization pattern can be used to determine qualitative and/or quantitative information about the expression of tumor associated antigen genes. The hybridization patterns of different samples can be compared with each other, or with a control sample, to identify differences between the patterns. The hybridization arrays can also be used to identify differential gene expression, in the analysis of diseased and normal tissue.
Antibody Production
Antibodies directed against cancer associated proteins include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, antibody fragments (e.g., Fab, Fab′, F(ab′)2, Fv, or hypervariable regions), bi-specific antibodies and humanized antibodies, methods of production of which are known in the art.
For the production of polyclonal antibodies, a host animal (e.g., rabbits, mice, rats, sheep, goats, and the like) can be immunized by injection with a tumor associated antigen, fragment, derivative or analog. Various adjuvants can be used to increase the immunological response, depending on the host species. Such adjuvants include, for example, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and other adjuvants, such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
Techniques for preparation of monoclonal antibodies include the original Kohler and Milstein hybridoma technique (see, e.g., Nature 256:495 97 (1975)), the trioma technique (see, e.g., Hagiwara and Yuasa, Hum. Antibodies Hybridomas 4:15 19 (1993); Hering et al., Biomed. Biochim. Acta 47:211 16 (1988)), the human B-cell hybridoma technique (see, e.g., Kozbor et al., Immunology Today 4:72 (1983)), and the EBV-hybridoma technique (see, e.g., Cole et al., In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96 (1985)). Human antibodies can be obtained using human hybridomas (see, e.g., Cote et al., Proc. Natl. Acad. Sci. USA 80:2026 30 (1983)) or by transforming human B cells with EBV virus in vitro (see, e.g., Cole et al., supra).
Chimeric antibodies are typically prepared by splicing the genes (of one species) for an antibody molecule specific for tumor associated antigen together with genes from another species of antibody molecule of appropriate biological activity. It can be desirable to transfer the antigen binding regions (e.g., Fab′, F(ab′)2, Fab, Fv, or hypervariable regions) of antibodies from one species into the framework of an antibody from another species by recombinant DNA techniques to produce a chimeric molecule. Methods for producing such molecules are described in, for example, U.S. Pat. Nos. 4,816,567; 4,816,397; 5,693,762, and 5,712,120. A human monoclonal antibody or portion(s) thereof can be identified by screening a human B-cell cDNA library for nucleic acid molecules that encode antibodies that specifically bind to a tumor associated antigen according to the method generally set forth by Huse et al. (Science 246:1275 81 (1989)). The nucleic acid molecule can then be cloned and amplified to obtain sequences that encode the antibody (or antigen-binding domain) of the desired specificity. Phage display technology offers another technique for selecting antibodies that bind to tumor associated antigens, fragments, derivatives or analogs thereof (see, e.g., International Patent Publications WO 91/17271 and WO 92/01047; Huse et al., supra.)
Techniques for the production of single chain antibodies are described for example in U.S. Pat. Nos. 4,946,778 and 5,969,108. A Fab expression library (see, e.g., Huse et al., supra) allows rapid and easy identification of monoclonal Fab fragments with the desired specificity for tumor associated antigens, fragments, derivatives, or analogs thereof.
F(ab′)2 antibody fragments can be produced by pepsin digestion of an antibody molecule. Fab′ fragments can be generated by reducing the disulfide bridges of a F(ab′)2 fragment, Fab and Fv fragments can be generated by treating an antibody molecule with papain and a reducing agent. Recombinant Fv fragments can also be produced in eukaryotic cells using, for example, as described in U.S. Pat. No. 5,965,405.
Bi-specific antibodies can be monoclonal antibodies that have binding specificities for at least two different antigens. For example, one of the binding specificities can be for a tumor associated antigen and the other one is for any other antigen. Alternatively, one specificity is for a first tumor associated antigen, while the other specificity is for a second, different tumor associated antigen. Methods for making bi-specific antibodies may be based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, Nature 305:537 39 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas produce a potential mixture of different antibody molecules, some of which have the desired bi-specific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion typically is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. The first heavy-chain constant region (CH1) containing the site necessary for light-chain binding is usually present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bi-specific antibodies see, for example, Suresh et al (Methods in Enzymology 121:210 (1986)).
To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which expresses the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al., supra.
Pharmaceutical Compositions and Agents
A pharmaceutical or antigen composition according to the invention comprises at least one protein selected from the group consisting of KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, LYZ, LOC442497; SLC3A2, DMBT1, NUCB1, GGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP and CLIC1, or an immunogenic fragment thereof.
The composition may be used in a therapeutic method for preventing or treating cancer, wherein an effective amount of the composition is administered to a subject in need thereof. A subject in need thereof may be for example, an individual in which colorectal polyps have been identified, and/or has been determined to have other risk factors for development of colorectal cancer. Accordingly, a method of preventing colorectal cancer may comprise administering an effective amount of a pharmaceutical composition comprising at least one protein highly expressed in polyps, for example any of CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1.
Further provided are pharmaceutical compositions comprising one or more reagents suitable for detecting the level of at least one protein selected from the group consisting of KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI, CISD1, LYZ, LOC442497; SLC3A2, DMBT1, NUCB1, UGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP and CLIC1.
As hereinbefore described, such reagents may comprise labeled antibodies, antibody mimetics or nucleic acids as the active ingredients. Advantageously such reagents are provided as a “cocktail” tailored to detect in vivo a particular panel of cancer associated proteins, or genes expressing same, Following administration, the localization of such reagents to specific body and organ regions may be detected by means of an external monitoring apparatus or system appropriate for detection and quantitation of the specific label incorporated into the reagents.
In yet other embodiments, pharmaceutical agents, in particular antibodies and an antibody mimetics, are provided, which may be used for targeted therapy of cancers expressing particular cancer associated proteins. Such, agents may advantageously incorporate a cytotoxic moiety, such as a plant toxin, a bacterial toxin, a radioactive atom or a chemotherapeutic agent. Further, the pharmaceutical agent may be in the form of a chemical conjugate or a fusion protein and provided in a suitably formulated pharmaceutical composition.
Plant toxins include for example, ricin, abrin, pokeweed antiviral protein, saporin, gelonin, and derivatives thereof. Bacterial toxins include for example, Pseudomonas exotoxin, diphtheria toxin and derivatives thereof, iodine-125 and radon. Radioactive atoms include for example, radium, cesium-137, iridium-192, americium-241 and gold-198.
Chemotherapeutic agents include, without limitation, alkylating agents, for example cyclophosphamide; nitrosoureas, for example carmustine and lomustine; antimetabolites, for example 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine and pemetrexed; anthracyclines, for example daunorubicin, doxorubicin respinomycin D and idarubicin; topoisomerase inhibitors, for example topotecan, irinotecan, etoposide and teniposide; mitotic inhibitors, for example paclitaxel, docetaxel, etoposide, vinblastine and vincristine; platinum based drugs, for example cisplatin, carboplatin and oxaliplatin; steroids for example hydrocortisone, dexamethasone, methylprednisolone and prednisolone; and anti-angiogenic agents, for example bevacizumab, thalidomide, dopamine and tetrathiomolybdate.
The pharmaceutical composition can be used to administer diagnostic reagents designed to detect levels of proteins disclosed herein, in particular KIAA0152 (SEQ ID NO:1), NAMPT (SEQ ID NO:2), PYCR1 (SEQ ID NO:3), GPX2 (SEQ ID NO:4), PRKDC (SEQ ID NO:5), ALDH18A1 (SEQ ID NO:6), OCIAD2 (SEQ ID NO:7), GCS1 (SEQ ID NO:8), GMDS (SEQ ID NO:9), ARF4 (SEQ ID NO:10), ARF5 (SEQ ID NO:11), LRPPRC (SEQ ID NO:12), CTNNB1 (SEQ ID NO:13), ARF3 (SEQ ID NO:14), GCN1L1 (SEQ ID NO:15), BDH1 (SEQ ID NO:16), RPL9 (SEQ ID NO:17), UGCGL1 (SEQ ID NO:18), FAM3D (SEQ ID NO:19) and CCT4 (SEQ ID NO:20), including antibodies, antibody mimetics or nucleic acids, or therapeutic reagents designed to exert a cytotoxic affect on their cancer targets. Therapeutic compositions for targeting tumors may include a reagent which specifically interacts with a protein selected from NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, KIAA0152, UGCGL1, FAM3D, CCT4, LYZ, LOC442497; SLC3A2, DMBT1, NUCB1, GGH, AGR3, TM9SF2, SYK, GCA, HDLBP, C1QBP, KIAA0152 and CLIC1.
The pharmaceutical composition may be prepared by suspending the desired reagent in any pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8. Other pharmaceutically acceptable carriers which are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
The pharmaceutical compositions may be prepared in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents. Sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
Pharmaceutical compositions that are useful in the methods of the invention may be administered, prepared, packaged, and/or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art.
Kits
The invention also includes kits for detecting, diagnosing, prognosing or staging a cancer or a tumor in a mammal. The cancer or tumor can be of any of the types described herein, and is preferably colorectal cancer. The kit comprises a container or a sample tube, or the like, for storing a sample of a cell, a population of cells, a tissue or a body fluid obtained from the mammal.
The kit also comprises one or more detection reagents selected from: an antibody or antibody mimetic which specifically bind with a cancer associated protein disclosed herein; a nucleic acid such as an oligonucleotide which specifically binds a nucleic acid (such as mRNA) encoding said cancer associated protein, and a PCR primer pair specific for a nucleic acid encoding said cancer associated protein. These detection reagents are as described herein. The kit comprises the one or more detection reagents in an amount effective to permit detection of the protein(s) of interest or a corresponding nucleic acid in the sample. Detection of the proteins or the nucleic acids is accomplished using any of the methods described herein or known to a skilled artisan for detecting a specific protein or specific nucleic acid molecule within a biological sample.
Reagents in the kit may be directed to a protein selected from KIAA0152, NAMPT, PYCR1, GPX2, PRKDC, ALDH18A1, OCIAD2, GCS1, GMDS, ARF4, ARF5, LRPPRC, CTNNB1, ARF3, GCN1L1, BDH1, RPL9, UGCGL1, FAM3D, CCT4, CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1. The kit may be intended for detecting pre-cancerous or early stage colorectal cancer and include reagents detecting any protein selected from the group consisting of CPT2, ARL1, PFKL, GOT2, AP1G1, STRBP, CLCA1, CYFIP1, COQ9, NDUFA9, ALDH7A1, HMGCS1, NNT, PRDX5, PCCB, COPZ1, BAX, ACAD9, UBXD8, HMGCS2, SLC25A3, SLC25A11, PDCD6, UCRC, DEFA6, DYNC1H1, HK1, CYFIP2, DCI and CISD1. In particular embodiments, the kit comprises a reagent suitable for detecting KIAA0152.
The kit also comprises at least one control biological sample, such as from non-diseased tissue, for comparison to a biological sample obtained from a subject under investigation. Control biological samples which correspond to samples from a cancer of a known stage or having a known prognosis, may also be included.
The kit also comprises an instructional material which directs the use of the reagents and the samples for the determining the amount and the location of the proteins or the nucleic acids in one or more cells of the sample. The instructional material also directs the correlation of the amount and the location of the protein or the nucleic acid in the cells of the sample with the diagnosis, prognosis and/or stage of a cancer or a tumor in the mammal.
As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which directs or dictates the use of the components of a kit for performing the function of a method of the invention described herein. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the composition or be shipped together with a container which contains the composition.
The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.
The following methods were used in the Examples.
Protein Extraction from Frozen Tissues
Surplus snap-frozen tumor and margin non-tumoral tissue were collected from patients undergoing surgery for colorectal cancer at either of two university teaching hospitals in Israel. Around 30 mg of frozen tissue were extracted from every tissue sample by mixing the tissue sample with in 0.5 ml of 8 M urea, 400 mM ammonium bicarbonate, and homogenized by high speed tissue homogenizer (Omni-TH) for 1 min, activated at full speed.
Identification of Proteins
1) Gel-slicing method. About 50 μg of the proteins extracted from the tumor tissues or the healthy tissues were resolved by 10% of SDS-PAGE. Each gel lane was subsequently stained with Coomassie blue. The stained gel lanes were cut into 12 slices. The gel pieces were de-stained by extensive washing with acetonitrile and ammonium bicarbonate. The proteins in each gel slice were proteolyzed inside the stained gel: stained slices were reduced with 10 mM DTT, incubated at 60° C. for 30 min, alkylated with 10 mM iodoacetamide, at room temperature for 30 min and digested with trypsin overnight at 37° C., using modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio. The tryptic peptides were analyzed by AC-MS/MS using the OrbitrapXL mass spectrometer (Thermo-Fisher) fitted with a capillary HPLC (Eksigent). The peptides were resolved on homemade capillary columns (75 micron ID) packed with reversed phase 3.5 micron beads Reprosil C18-Aqua, using a method described by (Ishihama, Rappsilber et al. 2002). The HPLC separations of the peptides were at flow rates of about 250 nanoliters per minute during 2 hrs and with 7-40% gradients of acetonitrile in the presence of 0.1% formic acid. The capillary columns were connected on line to the Orbitrap mass spectrometer through an electrospray interface. The mass spectrometer was operated in a data-dependent mode where the masses of the eluting peptides were measured at high accuracy in the Orbitrap part of the machine and the seven most intense masses, detected at each full MS spectra whose charge states were determined to be double and triple, were selected for fragmentation by CID in the linear trap at the subsequent seven CID fragmentations.
2) Multidimensional chromatography coupled with mass spectrometry. 50 μg of total protein extracts mixed with 100 μg in 8 M urea, 100 mM ammonium bicarbonate were treated for blocking all the sulfhydryls by first reducing disulfides by addition of 10 mM DTT, incubation at 60° C. for 30 min. The free disulfides were next blocked by carboxymethylation using 10 mM iodoacetamide and incubation at room temperature for 30 min. The denatured and carboxymethylated protein mixtures were diluted three-fold with water to reduce urea concentration to about 2M followed by digestion in solution, overnight at 37° C. using modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio. The resulting peptides from the trypsinized proteins were desalted with reversed-phase with a C18 tip disposable micro-columns (Harvard), eluted with 90% acetonitrile, dried and dissolved in 0.1% formic acid. The resulting peptides were resolved by multi-dimensional chromatography with on-line first dimension of strong cation exchange (SCX) chromatography 0.3×5 mm columns (LC Packings) using ten salt steps of 20, 40, 60, 80, 100, 120, 160, 200, 300 and 500 mM ammonium acetate in 5% acetonitrile with 0.1% acetic acid. The peptides from each increased salt elution from the SCX columns were transferred on-line to a C18 trap column (0.3×5 mm, LC-Packings), which was connected on-line to a Reprosil C18 homemade capillary columns (75 micron ID), resolved by 7-40% acetonitrile gradients, during 2 hrs, in the presence of 0.1% formic acid as described before for the gel-slicing method.
Resolving peptides by two-dimensional capillary chromatography has been described (Link, Eng et al. 1999), reviewed in (Link 2002). The mass spectrometry analysis was performed on-line as described above using data-dependent LC-MS/MS analysis with full MS in the Orbitrap and subsequent seven dependent ion-trap CID spectra of the most abundant doubly and triply charged peptides, detected in the full MS.
3) Isotope labeling peptides to enable quantitative analysis. Labeling tryptic peptides with light or heavy stable isotope reagents may rely on commercial reagents, reviewed in (Ong and Mann 2005; Regnier and Julka 2006). To facilitate the accurate comparison of the relative amounts of each of the proteins in the different samples, the mixture of peptides produced by the trypsinization of the entire in-solution protein proteolysis were covalently modified with light and heavy stable-isotope reagents. Reductive dimethylation labeling was done with heavy and light formaldehyde as described by (Hsu, Huang et al. 2003).
50 ug of proteolytic peptides were covalently modified with stable-isotope labeled (heavy and light formaldehyde) reagents (reductive dimethylation). The labeled peptides were resolved by multi-dimensional chromatography with on-line SCX column as described above.
Stable isotope labeling was also performed by iTRAQ (Ross, Huang et al. 2004) using a labeling kit that was purchased from Applied Biosystems and labeling was done according to the manufacturer's protocol. The labeled peptides were resolved by multi-dimensional chromatography with on-line SCX column as described above.
Bioinformatics
The mass spectrometry data of both the tryptic peptides obtained from proteolysis in the gel slices mentioned above and the tryptic peptides resolved by multidimensional chromatography were clustered and analyzed using the Pep-Miner software tool (Beer, Barnea et al. 2004). The search against the human part of the IPI database was done by using multiple search engines: Pep-Miner (Beer, Barnea et al. 2004), Mascot (Perkins, Pappin et al. 1999) and Sequest (Eng, McCormack et al. 1994). Both Mascot and Sequest were run together using the Protein Discoverer software tool (Thermo-Fisher).
Peptides were selected according to the following critera: 1) Mascot: ionScore>identityHigh and expValue<0.05 and deltaScore=0; 2) Sequest: ((xCorr>2 and chg</=2) or (xCorr>2.5 and chg>/=3)) and probability>15 and deltaScore=0 A peptide was used for the analysis if it was identified with the above criteria at least once for Sequest and once for Mascot, although not necessarily both in the same scan or run or patient.
The entire protein repertoires of healthy and diseased gastrointestinal tissues were analyzed from samples obtained from greater than 50 patients undergoing surgery for colorectal cancer.
The clinical characteristics of the patients studied are provided in Table 2.
TABLE 2
Patient
Age at
No.
Diagnosis
Gender
Appearance
Diagnosis
TNM
Stage
Grade
201
78
M
primary
Adeno-
T3N1M0
III
high
carcinoma
202
81
F
primary
Mucinous
T3N1
III
low
adeno-
carcinoma
203
85
M
primary
Adeno-
T3N0M0
II
high
carcinoma
205
71
F
primary
Adeno-
T3N1M0
III
high
carcinoma
207
81
F
primary
Adeno-
T3N2
III
high
carcinoma
208
78
M
primary
Adeno-
T3N2M1
IV
high
carcinoma
209
71
M
primary
Adeno-
T3N0M1
II
high
carcinoma
211
57
F
primary
Adeno-
T2N0M0
I
intermed
carcinoma
212
50
F
primary
Adeno-
T3N1M0
III
high
carcinoma
214
54
F
primary
Adeno-
T3N1M0
III
high
carcinoma
217
77
F
Local
Adeno-
T3N0M0
II
high
recurrance
carcinoma
218
79
M
primary
Adeno-
T3N2M1
IV
low
(T + P + N)
carcinoma
219
60
M
primary
Adeno-
T3N1M0
III
high
carcinoma
220
73
M
primary
Adeno-
T3N2M1
IV
high
carcinoma
Patient
Age at
Type of
Tumor
No.
Diagnosis
Gender
surgery
Diagnosis
location
Stage
Grade
J1 (T + N)
61
F
Lt colectomy
Adeno-
left colon
IV
intermed
carcinoma
J2 (T + N)
67
M
Lt colectomy
Adeno-
left colon
II
intermed
carcinoma
J3 (T + N)
70
M
Rt colectomy
Adeno-
right colon
II
intermed
carcinoma
J4 (T + N)
83
F
Sigmoidectomy
Adeno-
left colon
II
intermed
carcinoma
J5 (T + N)
41
M
Lt colectomy
Adeno-
left colon
I
intermed
carcinoma
J6 (T + N)
67
M
Lt colectomy
Adeno-
left colon
IV
intermed
carcinoma
J7 (T + N)
95
M
Rt colectomy
Adeno-
right colon
III
intermed
carcinoma
(T3N1)
J8 (T + N)
72
F
Rt colectomy
Adeno-
right + left
III
high
carcinoma
colon
T3N4
J9 (T + N)
56
F
Rt colectomy
Adeno-
right colon
II
intermed
carcinoma
T4N0M0
J10
64
M
Anterior
Adeno-
rectum
II
low
(T + N)
Resection
carcinoma
T4N0Mx
of rectum
J11
61
F
Rt colectomy
Adeno-
right colon
I
low
(T + N)
carcinoma
J12
61
M
colectomy
Adeno-
rectum
II
intermed
(T + N)
carcinoma
J13
60
M
Rt colectomy
Adeno-
right colon
I
intermed
(T + N)
carcinoma
J14
53
F
colectomy
Adeno-
left colon
II
intermed
(T + N)
carcinoma
J15
44
M
Lt colectomy
Colonic
left colon
(P + N)
polyps
J16 (P + N +
64
M
Rt colectomy
Tubulo-
right colon
Pbig)
villus
J17
81
M
Rt colectomy
Adeno-
right colon
II
intermed
(T + N)
carcinoma
J18
58
F
Rt colectomy
Adeno-
right colon
I
intermed
(P + N)
carcinoma
in TVA
J19
72
M
Rt colectomy
Adeno-
right colon
II
intermed
(T + N + P)
carcinoma
J20
60
F
Rt colectomy
Adeno-
left colon
II
intermed
(T + N)
carcinoma
J21
29
F
Rt colectomy
Adeno-
right colon
IV
low
(T + N)
carcinoma
J22
70
M
Rt colectomy
Adeno-
right colon
II
high
(T + N)
carcinoma
J23
92
F
Rt colectomy
Adeno-
right colon
III
intermed
(T + N)
carcinoma
J24
75
M
Rt colectomy
Villo-
right colon
(P + N)
tubulos
adenoma
J25
47
M
Rt colectomy
Adeno-
Rectac-
III
intermed
(T + N)
carcinoma
toigmoid
J26
77
M
Rt colectomy
Adeno-
right colon
III
intermed
(T + N)
carcinoma
J27
77
M
Lt colectomy
Adeno-
left colon
II
intermed
(T + N)
carcinoma
J28
77
M
ext Rt
Villo-
right colon
high
(Pbig + N +
colectomy
tubulos
P)
adenoma
J29
78
M
Lt colectomy
Adeno-
left colon
II
intermed
(T + N)
carcinoma
J30
67
F
Lt colectomy
Adeno-
right colon
III
intermed
(T + N)
carcinoma
J31
83
M
Sigmoidectomy
Adeno-
right colon
II
low
(T + N)
carcinoma
J32
65
F
Anterior
Adeno-
right colon
II
intermed
(T + N)
Resaction
carcinoma
J33
83
M
Rt colectomy
Adeno-
right colon
II
intermed
(N + P1 − 5)
carcinoma
J34
82
M
Rt colectomy
Adeno-
right colon
II
intermed-
(T + N)
carcinoma
high
J35
81
M
Gastrectomy
Adeno-
stomach
III
high
(T + N)
carcinoma
J36
62
M
Resaction
Adeno-
recto
III
intermed
(T + N)
carcinoma
signuoid
J37
55
M
Rt colectomy
Adeno-
III
intermed-
(T + N)
carcinoma
high
J38
80
M
Rt colectomy
Adeno-
right colon
III
intermed-
(T + N + P1 −
carcinoma
low
P2)
J39 (T + P)
62
M
Rt colectomy
Villo-
right colon
I
high
tubulos
adenoma
J40
52
M
Anterior
Adeno-
Sigmoid
III
intermed-
(T + N)
Resection
carcinoma
low
J41
87
M
Rt colectomy
Adeno-
right colon
III
intermed-
(T + N + P1 +
carcinoma
low
P2)
J42
87
M
Rt colectomy
Adeno-
I
intermed
(T + N + P1 +
carcinoma
P2)
T, tumor;
N, normal;
P, polyp;
high grade = poorly differentiated;
low grade = well differentiated;
intermediate grade = moderately well differentiated.
Table 3 lists proteins that were observed to be highly expressed in polyps, as well as in early and advanced stages of various colorectal cancers. As shown in Table 3 and in
TABLE 3
IPI
Acc. No.
nPeptides
Protein
nBigT
nSmallT
nBigP
nSmallP
IPI00022649
18
SLC12A2 Isoform 1 of
14
2
16
0
Solute carrier family 12
member 2
IPI00017526
3
S100P Protein S100-P
27
0
15
0
IPI00022255
16
OLFM4 Olfactomedin-4
21
2
15
0
precursor
IPI00024095
16
ANXA3 Annexin A3
19
1
15
0
IPI00555902
6
OCIAD2 Isoform 1 of
15
0
15
0
OCIA domain-
containing protein 2
IPI00376503
7
PYCR1 pyrroline-5-
20
0
14
0
carboxylate reductase 1
isoform 2
IPI00298176
7
GPX2 Glutathione
18
0
14
0
peroxidase 2
IPI00296337
55
PRKDC Isoform 1 of
16
2
14
0
DNA-dependent protein
kinase catalytic subunit
IPI00215919
9
ARF5 ADP-ribosylation
11
2
14
0
factor 5
IPI00619903
21
UGCGL1 UDP-
9
2
14
0
glucose:glycoprotein
glucosyltransferase 1
precursor
IPI00018873
18
NAMPT Isoform 1 of
17
2
13
0
Nicotinamide
phosphoribosyltransferase
IPI00008982
16
ALDH18A1 Isoform
16
2
13
0
Long of Delta-1-
pyrroline-5-carboxylate
synthetase
IPI00328170
6
GCS1 Mannosyl-
15
0
13
0
oligosaccharide
glucosidase
IPI00216057
8
SORD Sorbitol
14
2
13
0
dehydrogenase
IPI00030207
16
GMDS GDP-mannose
13
2
13
0
4,6 dehydratase
IPI00215918
10
ARF4 ADP-ribosylation
12
2
13
0
factor 4
IPI00783271
45
LRPPRC Leucine-rich
11
3
13
0
PPR motif-containing
protein, mitochondrial
precursor
IPI00017292
23
CTNNB1 Isoform 1 of
11
2
13
0
Catenin beta-1
IPI00215917
10
ARF3 ADP-ribosylation
11
2
13
0
factor 3
IPI00001159
18
GCN1L1 Translational
11
1
13
0
activator GCN1
IPI00025341
10
BDH1 D-beta-
10
4
13
0
hydroxybutyrate
dehydrogenase,
mitochondrial precursor
IPI00031691
9
RPL9 60S ribosomal
10
1
13
0
protein L9
IPI00029046
7
KIAA0152
10
0
13
0
Uncharacterized protein
KIAA0152 precursor
IPI00060143
5
FAM3D Protein
9
4
13
0
FAM3D precursor
IPI00302927
18
CCT4 T-complex
8
1
13
0
protein 1 subunit delta
IPI00643623
6
LCN2 Lipocalin 2
23
0
12
0
IPI00217223
13
PAICS Multifunctional
17
2
12
0
protein ADE2
IPI00012501
4
REG4 Isoform 1 of
16
0
12
0
Regenerating islet-
derived protein 4
precursor
IPI00793443
14
IPO5 RAN binding
15
1
12
0
protein 5
IPI00025273
15
GART Isoform Long of
15
1
12
0
Trifunctional purine
biosynthetic protein
adenosine-3
IPI00646687
19
POF1B Isoform 2 of
14
2
12
0
Protein POF1B
IPI00023728
8
GGH Gamma-glutamyl
14
1
12
0
hydrolase precursor
IPI00306301
13
PDHA1 Mitochondrial
13
3
12
1
PDHA1
IPI00006379
12
NOP5/NOP58 Nucleolar
13
0
12
0
protein 5
IPI00171692
7
ABHD11 Isoform 1 of
13
2
12
0
Abhydrolase domain-
containing protein 11
IPI00329719
25
MYO1D Isoform 1 of
12
4
12
0
Myosin-Id
IPI00216225
16
ITGA6 Isoform Alpha-
12
2
12
0
6X1X2A of Integrin
alpha-6 precursor
IPI00103994
15
LARS Leucyl-tRNA
12
2
12
0
synthetase, cytoplasmic
IPI00005198
8
ILF2 Interleukin
12
1
12
0
enhancer-binding factor 2
IPI00783625
5
SERPINB5 Serpin B5
12
0
12
0
precursor
IPI00789324
22
JUP JUP protein
11
2
12
0
IPI00871852
21
EIF4A1 46 kDa protein
11
2
12
0
IPI00646493
28
COPA coatomer protein
11
2
12
0
complex, subunit alpha
isoform 1
IPI00783982
20
COPG Coatomer
11
2
12
0
subunit gamma
IPI00295851
23
COPB1 Coatomer
11
2
12
0
subunit beta
IPI00007928
17
PRPF8 Pre-mRNA-
11
1
12
0
processing-splicing
factor 8
IPI00008164
17
PREP Prolyl
11
1
12
0
endopeptidase
IPI00848161
15
BAT1 Isoform 1 of
10
1
12
0
Spliceosome RNA
helicase BAT1
IPI00844578
26
DHX9 ATP-dependent
10
2
12
0
RNA helicase A
IPI00383680
19
RPN2 Ribophorin II
10
2
12
0
IPI00011253
14
RPS3 40S ribosomal
10
1
12
0
protein S3
IPI00300371
13
SF3B3 Isoform 1 of
10
0
12
0
Splicing factor 3B
subunit 3
IPI00893013
11
XPO1 123 kDa protein
10
1
12
0
IPI00790342
11
RPL6 60S ribosomal
10
2
12
0
protein L6
IPI00011511
7
CECR5 Isoform 2 of Cat
10
4
12
0
eye syndrome critical
region protein 5
precursor
IPI00023542
4
TMED9 transmembrane
10
0
12
0
emp24 protein transport
domain containing 9
IPI00073772
8
FBP1 Fructose-1,6-
9
4
12
0
bisphosphatase 1
IPI00893057
16
PDXDC1 87 kDa
8
3
12
0
protein
IPI00029012
15
EIF3A Eukaryotic
8
1
12
0
translation initiation
factor 3 subunit A
IPI00003833
10
MTCH2 Mitochondrial
8
2
12
0
carrier homolog 2
IPI00792875
3
SERPINB5 14 kDa
8
2
12
0
protein
IPI00306960
9
NARS Asparaginyl-
7
1
12
0
tRNA synthetase,
cytoplasmic
IPI00012912
12
CPT2 Carnitine O-
6
5
12
0
palmitoyltransferase 2,
mitochondrial precursor
IPI00219518
4
ARL1 ADP-ribosylation
6
1
12
0
factor-like protein 1
IPI00236556
23
MPO Isoform H7 of
24
2
11
0
Myeloperoxidase
precursor
IPI00218993
20
HSPH1 Isoform Beta of
21
3
11
0
Heat shock protein 105 kDa
IPI00477179
8
DDX21 Isoform 2 of
17
1
11
0
Nucleolar RNA helicase 2
IPI00215801
10
RBM39 Isoform 2 of
15
1
11
0
RNA-binding protein 39
IPI00554788
32
KRT18 Keratin, type I
13
2
11
0
cytoskeletal 18
IPI00893918
14
VARS Valyl-tRNA
13
2
11
0
synthetase
IPI00877938
11
IARS isoleucyl-tRNA
13
1
11
0
synthetase
IPI00641181
2
MARCKSL1
13
0
11
0
MARCKS-related
protein
IPI00401990
17
ACSL5 acyl-CoA
12
2
11
0
synthetase long-chain
family member 5
isoform a
IPI00396435
16
DHX15 Putative pre-
12
2
11
0
mRNA-splicing factor
ATP-dependent RNA
helicase DHX15
IPI00026089
14
SF3B1 Splicing factor
12
1
11
0
3B subunit 1
IPI00005158
14
LONP1 Lon protease
12
2
11
0
homolog, mitochondrial
precursor
IPI00479262
12
EIF4G1 Isoform B of
12
1
11
0
Eukaryotic translation
initiation factor 4
gamma 1
IPI00644431
12
DDX39 ATP-dependent
11
2
11
0
RNA helicase DDX39
IPI00028931
26
DSG2 Desmoglein-2
11
4
11
0
precursor
IPI00013452
20
EPRS Bifunctional
11
3
11
0
aminoacyl-tRNA
synthetase
IPI00418313
17
ILF3 Isoform 4 of
11
3
11
0
Interleukin enhancer-
binding factor 3
IPI00017376
7
SEC23B Protein
11
2
11
0
transport protein Sec23B
IPI00297084
8
DDOST dolichyl-
11
1
11
0
diphosphooligosaccharide-
protein
glycosyltransferase
precursor
IPI00411937
5
NOL5A Nucleolar
11
1
11
1
protein 5A
IPI00221091
5
RPS15A 40S ribosomal
11
0
11
0
protein S15a
IPI00015872
3
TSPAN8 Tetraspanin-8
11
7
11
0
IPI00784044
11
MCCC2 Isoform 1 of
10
3
11
0
Methylcrotonoyl-CoA
carboxylase beta chain,
mitochondrial precursor
IPI00013485
10
RPS2 40S ribosomal
10
2
11
0
protein S2
IPI00006684
7
API5 58 kDa protein
10
2
11
0
IPI00028004
5
PSMB3 Proteasome
10
2
11
0
subunit beta type-3
IPI00019385
5
SSR4 Translocon-
10
2
11
0
associated protein
subunit delta precursor
IPI00016608
5
TMED2
10
1
11
0
Transmembrane emp24
domain-containing
protein 2 precursor
IPI00329791
5
DDX46 cDNA
10
0
11
0
FLJ78679, highly
similar to Homo sapiens
DEAD (Asp-Glu-Ala-
Asp) box polypeptide 46
(DDX46), mRNA
IPI00215948
35
CTNNA1 Isoform 1 of
9
3
11
0
Catenin alpha-1
IPI00217952
29
GFPT1 Isoform 1 of
9
2
11
0
Glucosamine--fructose-
6-phosphate
aminotransferase
[isomerizing] 1
IPI00220834
18
XRCC5 ATP-dependent
9
1
11
0
DNA helicase 2 subunit 2
IPI00514622
3
RANBP6 Ran-binding
9
0
11
0
protein 6
IPI00027252
16
PHB2 Prohibitin-2
9
2
11
0
IPI00020672
13
DPP3; BBS1 Isoform 1
9
3
11
0
of Dipeptidyl-peptidase 3
IPI00017895
13
GPD2 Isoform 1 of
9
2
11
0
Glycerol-3-phosphate
dehydrogenase,
mitochondrial precursor
IPI00872756
10
ASL 58 kDa protein
9
4
11
0
IPI00329598
8
HSD17B11 Estradiol
9
3
11
1
17-beta-dehydrogenase
11 precursor
IPI00013296
6
RPS18; LOC100130553
9
1
11
0
40S ribosomal protein
S18
IPI00477831
14
ERAP1 Isoform 1 of
8
2
11
0
Endoplasmic reticulum
aminopeptidase 1
IPI00303207
7
ABCE1 ATP-binding
8
0
11
0
cassette sub-family E
member 1
IPI00847192
6
RPS9 protein
8
1
11
0
(Fragment)
IPI00441344
5
GLB1 Isoform 1 of
8
1
11
0
Beta-galactosidase
precursor
IPI00418497
3
TIMM50 Isoform 2 of
8
0
11
0
Import inner membrane
translocase subunit
TIM50, mitochondrial
precursor
IPI00646182
40
ATP1A1 ATPase,
7
3
11
0
Na+/K+ transporting,
alpha 1 polypeptide
IPI00009634
20
SQRDL Sulfide:quinone
7
3
11
0
oxidoreductase,
mitochondrial precursor
IPI00304171
12
H2AFY Isoform 2 of
7
1
11
0
Core histone macro-
H2A.1
IPI00022887
6
ERGIC1 Isoform 1 of
7
5
11
0
Endoplasmic reticulum-
Golgi intermediate
compartment protein 1
IPI00171626
2
LPCAT1 1-
7
0
11
0
acylglycerophosphocholine
O-acyltransferase 1
IPI00332371
20
PFKL Isoform 1 of 6-
6
2
11
0
phosphofructokinase,
liver type
IPI00018206
15
GOT2 Aspartate
6
3
11
0
aminotransferase,
mitochondrial precursor
IPI00643591
5
AP1G1 AP-1 complex
6
1
11
0
subunit gamma-1
IPI00413860
2
STRBP Isoform 2 of
6
0
11
0
Spermatid perinuclear
RNA-binding protein
IPI00014625
42
CLCA1 Calcium-
4
13
11
1
activated chloride
channel regulator 1
precursor
IPI00644231
14
CYFIP1 Isoform 1 of
4
2
11
0
Cytoplasmic FMR1-
interacting protein 1
IPI00470631
4
COQ9 Isoform 1 of
4
3
11
0
Ubiquinone biosynthesis
protein COQ9,
mitochondrial precursor
IPI00003968
9
NDUFA9 NADH
3
4
11
0
dehydrogenase
[ubiquinone] 1 alpha
subcomplex subunit 9,
mitochondrial precursor
IPI00221234
14
ALDH7A1 Similar to
2
5
11
0
Antiquitin
IPI00007047
6
S100A8 Protein S100-
29
0
10
0
A8
IPI00027462
6
S100A9 Protein S100-
27
1
10
0
A9
IPI00021700
8
PCNA Proliferating cell
18
0
10
0
nuclear antigen
IPI00297579
7
CBX3; LOC653972
18
1
10
0
Chromobox protein
homolog 3
IPI00642046
4
RSL1D1 RSL1D1
18
1
10
0
protein
IPI00002520
14
SHMT2 Serine
17
2
10
0
hydroxymethyltransferase,
mitochondrial
precursor
IPI00219871
2
LSM8 U6 snRNA-
16
2
10
1
associated Sm-like
protein LSm8
IPI00030275
17
TRAP1 Heat shock
15
2
10
0
protein 75 kDa,
mitochondrial precursor
IPI00893035
8
CAD Putative
15
2
10
0
uncharacterized protein
CAD
IPI00553131
9
GALE UDP-glucose 4-
14
2
10
0
epimerase
IPI00217477
3
HMGB3 High mobility
14
3
10
0
group protein B3
IPI00295992
9
ATAD3A Isoform 2 of
13
2
10
0
ATPase family AAA
domain-containing
protein 3A
IPI00646721
9
USP7 Ubiquitin
13
9
10
0
carboxyl-terminal
hydrolase
IPI00465044
8
RCC2 Protein RCC2
13
1
10
0
IPI00420014
17
ASCC3L1 Isoform 1 of
12
0
10
0
U5 small nuclear
ribonucleoprotein 200 kDa
helicase
IPI00027444
16
SERPINB1 Leukocyte
12
2
10
0
elastase inhibitor
IPI00216308
14
VDAC1 Voltage-
12
2
10
0
dependent anion-
selective channel protein 1
IPI00334175
14
PTBP1 Isoform 2 of
12
2
10
0
Polypyrimidine tract-
binding protein 1
IPI00218466
8
SEC61A1 Isoform 1 of
12
1
10
0
Protein transport protein
Sec61 subunit alpha
isoform 1
IPI00019912
24
HSD17B4 Peroxisomal
11
1
10
0
multifunctional enzyme
type 2
IPI00002557
4
COPG2 Coatomer
11
3
10
0
subunit gamma-2
IPI00449049
16
PARP1 Poly [ADP-
11
1
10
0
ribose] polymerase 1
IPI00014238
10
KARS Lysyl-tRNA
11
2
10
0
synthetase
IPI00008530
10
RPLP0 60S acidic
11
2
10
0
ribosomal protein P0
IPI00009950
8
LMAN2 Vesicular
11
2
10
0
integral-membrane
protein VIP36 precursor
IPI00026202
6
RPL18A 60S ribosomal
11
2
10
0
protein L18a
IPI00018597
3
SYK Isoform Long of
11
0
10
0
Tyrosine-protein kinase
SYK
IPI00216951
21
DARS Aspartyl-tRNA
10
2
10
0
synthetase, cytoplasmic
IPI00747497
14
EEF1G 50 kDa protein
10
2
10
0
IPI00032038
14
CPT1A Isoform 1 of
10
7
10
0
Carnitine O-
palmitoyltransferase I,
liver isoform
IPI00297492
10
STT3A Dolichyl-
10
1
10
0
diphosphooligosaccharide--
protein
glycosyltransferase
subunit STT3A
IPI00549672
8
PSMD13 HSPC027
10
0
10
0
IPI00221089
7
RPS13 40S ribosomal
10
1
10
0
protein S13
IPI00152377
7
STT3B Dolichyl-
10
0
10
0
diphosphooligosaccharide--
protein
glycosyltransferase
subunit STT3B
IPI00028055
6
TMED10
10
2
10
0
Transmembrane emp24
domain-containing
protein 10 precursor
IPI00032139
3
SERPINB9 Serpin B9
10
1
10
0
IPI00290089
28
CDH17 Cadherin-17
9
5
10
0
precursor
IPI00797038
21
PCK2 mitochondrial
9
2
10
0
phosphoenolpyruvate
carboxykinase 2 isoform
1 precursor
IPI00004860
18
RARS Isoform
9
2
10
0
Complexed of Arginyl-
tRNA synthetase,
cytoplasmic
IPI00026665
13
QARS Glutaminyl-
9
1
10
0
tRNA synthetase
IPI00017283
10
IARS2 Isoleucyl-tRNA
9
2
10
0
synthetase,
mitochondrial precursor
IPI00030847
7
TM9SF3
9
1
10
0
Transmembrane 9
superfamily member 3
precursor
IPI00014361
5
TSTA3 GDP-L-fucose
9
0
10
0
synthetase
IPI00008298
1
DEFA5 Defensin-5
9
0
10
0
precursor
IPI00033022
15
DNM2 Isoform 1 of
8
1
10
0
Dynamin-2
IPI00220847
19
ITGB4 Isoform Beta-4D
8
5
10
0
of Integrin beta-4
precursor
IPI00847318
15
PKP2 plakophilin 2
8
5
10
0
isoform 2a
IPI00215911
11
APEX1 DNA-(apurinic
8
1
10
0
or apyrimidinic site)
lyase
IPI00874185
10
HIBCH 46 kDa protein
8
5
10
0
IPI00182533
6
RPL28 60S ribosomal
8
6
10
0
protein L28
IPI00296909
6
PARP4 Poly [ADP-
8
2
10
0
ribose] polymerase 4
IPI00023876
5
CASP6 Isoform Alpha
8
3
10
0
of Caspase-6 precursor
IPI00744194
5
Similar to
7
4
10
0
Sodium/potassium-
transporting ATPase
alpha-1 chain precursor
IPI00302925
28
CCT8 59 kDa protein
7
2
10
0
IPI00100160
19
CAND1 Isoform 1 of
7
1
10
0
Cullin-associated
NEDD8-dissociated
protein 1
IPI00337494
13
SLC25A24 Isoform 1 of
7
3
10
0
Calcium-binding
mitochondrial carrier
protein SCaMC-1
IPI00747849
6
ATP1B1 Isoform 1 of
7
3
10
0
Sodium/potassium-
transporting ATPase
subunit beta-1
IPI00008433
5
RPS5 40S ribosomal
7
2
10
0
protein S5
IPI00008475
3
HMGCS1
6
4
10
0
Hydroxymethylglutaryl-
CoA synthase,
cytoplasmic
IPI00337541
18
NNT NAD(P)
6
2
10
0
transhydrogenase,
mitochondrial precursor
IPI00876999
12
PRDX5 Uncharacterized
6
2
10
0
protein PRDX5
(Fragment)
IPI00007247
12
PCCB Propionyl-CoA
6
5
10
0
carboxylase beta chain,
mitochondrial precursor
IPI00032851
5
COPZ1 Coatomer
6
4
10
0
subunit zeta-1
IPI00845474
5
BAX BCL2-associated
6
2
10
0
X protein isoform sigma
IPI00152981
3
ACAD9 Acyl-CoA
6
0
10
0
dehydrogenase family
member 9,
mitochondrial precursor
IPI00172656
1
UBXD8 UBX domain-
6
1
10
0
containing protein 8
IPI00008934
20
HMGCS2
5
12
10
0
Hydroxymethylglutaryl-
CoA synthase,
mitochondrial precursor
IPI00790115
11
SLC25A3 cDNA
5
1
10
0
FLJ90278 fis, clone
NT2RP1000325, highly
similar to Phosphate
carrier protein,
mitochondrialprecursor
IPI00219729
9
SLC25A11
5
4
10
0
Mitochondrial 2-
oxoglutarate/malate
carrier protein
IPI00025277
5
PDCD6 Programmed
5
0
10
0
cell death protein 6
IPI00554701
2
UCRC Cytochrome b-c1
4
0
10
0
complex subunit 9
IPI00008301
2
DEFA6 Defensin-6
4
0
10
0
precursor
IPI00456969
88
DYNC1H1 Cytoplasmic
3
1
10
0
dynein 1 heavy chain 1
IPI00220663
17
HK1 Isoform 2 of
3
1
10
0
Hexokinase-1
IPI00719600
7
CYFIP2 Isoform 2 of
3
2
10
0
Cytoplasmic FMR1-
interacting protein 2
IPI00398758
5
DCI Isoform 2 of 3,2-
3
5
10
0
trans-enoyl-CoA
isomerase,
mitochondrial precursor
IPI00020510
3
CISD1 CDGSH iron
1
1
10
0
sulfur domain-
containing protein 1
IPI00294443
2
CLIC5 Isoform 1 of
21
0
9
0
Chloride intracellular
channel protein 5
IPI00031564
7
C7orf24
17
1
9
0
Uncharacterized protein
C7orf24
IPI00871140
10
NP Purine nucleoside
16
1
9
0
phosphorylase
IPI00291510
9
IMPDH2 Inosine-5′-
15
1
9
0
monophosphate
dehydrogenase 2
IPI00792186
5
ABCF1 ATP-binding
15
0
9
0
cassette, sub-family F
(GCN20), member 1
IPI00029744
4
SSBP1 Single-stranded
15
3
9
0
DNA-binding protein,
mitochondrial precursor
IPI00166680
2
MINK1 Isoform 3 of
15
1
9
0
Misshapen-like kinase 1
IPI00152441
3
HM13 Isoform 1 of
14
0
9
0
Minor
histocompatibility
antigen H13
IPI00303318
8
FAM49B Protein
13
2
9
0
FAM49B
IPI00643166
4
PGM3 Isoform 2 of
13
0
9
0
Phosphoacetylglucosamine
mutase
IPI00641950
14
GNB2L1 Lung cancer
12
2
9
0
oncogene 7
IPI00293464
11
DDB1 DNA damage-
12
1
9
0
binding protein 1
IPI00783097
10
GARS Glycyl-tRNA
12
2
9
0
synthetase
IPI00219617
4
PRPS2 Isoform 1 of
12
1
9
0
Ribose-phosphate
pyrophosphokinase 2
IPI00018415
7
TM9SF2
12
1
9
0
Transmembrane 9
superfamily member 2
precursor
IPI00550032
4
LOC653232; RPL15
12
0
9
0
Ribosomal protein L15
pseudogene 3
IPI00013933
65
DSP Isoform DPI of
11
2
9
0
Desmoplakin
IPI00009032
15
SSB Lupus La protein
11
1
9
0
IPI00792100
10
C14orf166 CLE
11
1
9
0
IPI00010491
3
RAB27B Ras-related
11
2
9
0
protein Rab-27B
IPI00302850
3
SNRPD1 Small nuclear
11
1
9
0
ribonucleoprotein Sm
D1
IPI00644712
20
XRCC6 ATP-dependent
10
1
9
0
DNA helicase 2 subunit 1
IPI00025874
20
RPN1 Dolichyl-
10
2
9
0
diphosphooligosaccharide--
protein
glycosyltransferase 67 kDa
subunit precursor
IPI00744889
9
CDH1 E-cadherin
10
2
9
0
IPI00002372
8
ABCD3 Isoform 1 of
10
4
9
0
ATP-binding cassette
sub-family D member 3
IPI00289601
4
HDAC2 histone
10
2
9
0
deacetylase 2
IPI00328867
5
SRC Isoform 2 of Proto-
10
0
9
0
oncogene tyrosine-
protein kinase Src
IPI00872474
3
LYN LYN protein
10
0
9
0
(Fragment)
IPI00031804
8
VDAC3 Isoform 1 of
10
2
9
0
Voltage-dependent
anion-selective channel
protein 3
IPI00654777
7
EIF3F Eukaryotic
10
0
9
0
translation initiation
factor 3 subunit 5
IPI00440703
6
GSTK1 GSTK1 protein
10
3
9
0
IPI00885106
3
TM9SF4 Isoform 2 of
10
0
9
0
Transmembrane 9
superfamily member 4
precursor
IPI00029267
2
SNRPB2 U2 small
10
2
9
0
nuclear
ribonucleoprotein B″
IPI00414717
12
GLG1 golgi apparatus
9
1
9
0
protein 1
IPI00216293
11
TST Thiosulfate
9
6
9
0
sulfurtransferase
IPI00029629
11
TRIM25 Tripartite
9
2
9
0
motif-containing protein
25
IPI00219147
4
CSDA Isoform 2 of
9
1
9
0
DNA-binding protein A
IPI00219953
9
CMPK1 cytidine
9
3
9
0
monophosphate (UMP-
CMP) kinase 1,
cytosolic
IPI00827508
7
RPL10A 25 kDa protein
9
2
9
0
IPI00014053
6
TOMM40 Isoform 1 of
9
0
9
0
Mitochondrial import
receptor subunit TOM40
homolog
IPI00299048
20
IQGAP2 Isoform 1 of
8
2
9
0
Ras GTPase-activating-
like protein IQGAP2
IPI00242956
70
FCGBP IgGFc-binding
8
9
9
2
protein precursor
IPI00291467
18
SLC25A6 ADP/ATP
8
2
9
0
translocase 3
IPI00290566
22
TCP1 T-complex
8
2
9
0
protein 1 subunit alpha
IPI00470502
14
PPA2 Isoform 2 of
8
1
9
0
Inorganic
pyrophosphatase 2,
mitochondrial precursor
IPI00003482
12
DECR1 2,4-dienoyl-
8
4
9
0
CoA reductase,
mitochondrial precursor
IPI00456750
10
FAM129B Niban-like
8
3
9
0
protein 1
IPI00303158
10
CMAS Isoform 1 of N-
8
2
9
0
acylneuraminate
cytidylyltransferase
IPI00001466
8
EML4 Echinoderm
8
2
9
0
microtubule-associated
protein-like 4
IPI00105598
7
PSMD11 Proteasome
8
1
9
0
26S non-ATPase subunit
11 variant (Fragment)
IPI00011916
4
JTV1 Multisynthetase
8
1
9
0
complex auxiliary
component p38
IPI00100460
3
DARS2 Aspartyl-tRNA
8
1
9
0
synthetase,
mitochondrial precursor
IPI00030706
3
AHSA1 Activator of 90 kDa
8
1
9
0
heat shock protein
ATPase homolog 1
IPI00874145
3
DKC1 Uncharacterized
8
0
9
0
protein DKC1
(Fragment)
IPI00177817
24
ATP2A2 Isoform
7
2
9
0
SERCA2A of
Sarcoplasmic/endoplasmic
reticulum calcium
ATPase 2
IPI00409717
13
EIF4A2 Isoform 2 of
7
1
9
0
Eukaryotic initiation
factor 4A-II
IPI00719752
15
EIF3B Isoform 2 of
7
2
9
0
Eukaryotic translation
initiation factor 3
subunit B
IPI00001091
12
AFG3L2 AFG3-like
7
7
9
0
protein 2
IPI00026530
10
LMAN1 Protein
7
2
9
0
ERGIC-53 precursor
IPI00007676
9
HSD17B12 Estradiol
7
2
9
0
17-beta-dehydrogenase
12
IPI00291930
8
CLINT1 Isoform 1 of
7
2
9
0
Clathrin interactor 1
IPI00293853
7
GPA33 Cell surface
7
6
9
0
A33 antigen precursor
IPI00797738
5
COX6B1 12 kDa
7
2
9
0
protein
IPI00885058
3
MBOAT7 Isoform 2 of
7
5
9
0
Membrane-bound O-
acyltransferase domain-
containing protein 7
IPI00455383
68
CLTC Isoform 2 of
6
2
9
0
Clathrin heavy chain 1
IPI00102864
12
HK2 Hexokinase-2
6
2
9
0
IPI00018931
16
VPS35 Vacuolar protein
6
0
9
1
sorting-associated
protein 35
IPI00010157
8
MAT2A S-
6
1
9
1
adenosylmethionine
synthetase isoform type-2
IPI00328715
6
MTDH Protein LYRIC
6
1
9
0
IPI00171573
5
CCDC109A Isoform 1
6
0
9
0
of Coiled-coil domain-
containing protein 109A
IPI00411426
5
VPS26A Vacuolar
6
1
9
0
protein sorting-
associated protein 26A
IPI00017767
2
MGST2 Microsomal
6
2
9
0
glutathione S-transferase 2
IPI00556311
2
DUOX2 Dual oxidase 2
6
0
9
0
variant (Fragment)
IPI00011201
10
ME2 NAD-dependent
5
3
9
0
malic enzyme,
mitochondrial precursor
IPI00329672
5
MYO1E Myosin-Ie
5
1
9
0
IPI00020928
4
TFAM Transcription
5
4
9
0
factor A, mitochondrial
precursor
IPI00009104
12
RUVBL2 RuvB-like 2
4
0
9
0
IPI00744115
12
PCCA propionyl-
4
3
9
0
Coenzyme A
carboxylase, alpha
polypeptide precursor
IPI00219029
10
GOT1 Aspartate
4
3
9
0
aminotransferase,
cytoplasmic
IPI00029264
7
CYC1 Cytochrome c1,
4
2
9
0
heme protein,
mitochondrial precursor
IPI00027448
5
ATP5L ATP synthase
4
2
9
0
subunit g, mitochondrial
IPI00215920
2
ARF6 ADP-ribosylation
4
0
9
0
factor 6
IPI00220244
1
TRIM23 Isoform Beta
4
0
9
0
of GTP-binding protein
ARD-1
IPI00006674
7
ABCC3 Isoform 3 of
3
4
9
0
Canalicular
multispecific organic
anion transporter 2
IPI00843876
7
TNPO1 Transportin-1
3
0
9
0
IPI00023001
3
C3orf28 E2-induced
3
5
9
0
gene 5 protein
IPI00019038
4
LYZ Lysozyme C
17
2
8
0
precursor
IPI00022462
14
TFRC Transferrin
16
0
8
0
receptor protein 1
IPI00554722
7
LOC442497; SLC3A2
16
1
8
0
solute carrier family 3
(activators of dibasic
and neutral amino acid
transport), member 2
isoform e
IPI00010341
6
PRG2 Bone marrow
16
7
8
0
proteoglycan precursor
IPI00010320
2
CBX1 Chromobox
16
1
8
0
protein homolog 1
IPI00006690
27
EPX Eosinophil
15
8
8
0
peroxidase precursor
IPI00099110
4
DMBT1 Isoform 1 of
15
0
8
1
Deleted in malignant
brain tumors 1 protein
precursor
IPI00008240
8
MARS Methionyl-tRNA
14
1
8
0
synthetase, cytoplasmic
IPI00414676
41
HSP90AB1 Heat shock
13
2
8
0
protein HSP 90-beta
IPI00140420
27
SND1 Staphylococcal
13
2
8
0
nuclease domain-
containing protein 1
IPI00807557
14
PA2G4 PA2G4 protein
13
2
8
0
(Fragment)
IPI00215879
6
SFRS6 Isoform SRP55-
13
1
8
0
3 of Splicing factor,
arginine/serine-rich 6
IPI00301936
8
ELAVL1 ELAV-like
13
1
8
0
protein 1
IPI00025039
7
FBL rRNA 2′-O-
13
2
8
0
methyltransferase
fibrillarin
IPI00025427
4
RNASE3 Eosinophil
13
6
8
1
cationic protein
precursor
IPI00873179
4
CLC Uncharacterized
13
6
8
0
protein CLC (Fragment)
IPI00655650
3
LOC728937; RPS26 40S
13
1
8
0
ribosomal protein S26
IPI00888987
2
LOC345630 similar to
13
1
8
0
hCG1641252
IPI00026952
13
PKP3 Plakophilin-3
12
1
8
0
IPI00009328
12
EIF4A3 Eukaryotic
12
2
8
0
initiation factor 4A-III
IPI00759824
6
ANP32B Isoform 2 of
12
2
8
1
Acidic leucine-rich
nuclear phosphoprotein
32 family member B
IPI00029048
9
TTLL12 Tubulin--
12
3
8
0
tyrosine ligase-like
protein 12
IPI00029764
6
SF3A3 Splicing factor
12
1
8
0
3A subunit 3
IPI00303954
4
CYB5B cytochrome b5
12
2
8
0
outer mitochondrial
membrane precursor
IPI00007402
4
IPO7 Importin-7
12
0
8
0
IPI00012382
2
SNRPA U1 small
12
2
8
0
nuclear
ribonucleoprotein A
IPI00446377
2
ENG cDNA FLJ41744
12
0
8
0
fis, clone
HSYRA2005496, highly
similar to ENDOGLIN
IPI00186290
45
EEF2 Elongation factor 2
11
2
8
0
IPI00304596
18
NONO Non-POU
11
2
8
0
domain-containing
octamer-binding protein
IPI00220219
17
COPB2 Coatomer
11
2
8
0
subunit beta′
IPI00003918
16
RPL4 60S ribosomal
11
2
8
0
protein L4
IPI00020632
14
ASS1 Argininosuccinate
11
3
8
0
synthase
IPI00396661
5
CYP2S1 Isoform 1 of
11
0
8
0
Cytochrome P450 2S1
IPI00005589
5
hCG_18290
11
1
8
0
Uncharacterized protein
ENSP00000275524
IPI00178440
4
EEF1B2 Elongation
11
1
8
0
factor 1-beta
IPI00894416
4
BZW2 47 kDa protein
11
0
8
0
IPI00793862
3
SHMT1 Serine
11
0
8
0
hydroxymethyltransferase
IPI00029750
3
RPS24 Isoform 1 of 40S
11
1
8
0
ribosomal protein S24
IPI00873680
3
EIF4E Uncharacterized
11
2
8
0
protein EIF4E
(Fragment)
IPI00784154
45
HSPD1 60 kDa heat
10
3
8
0
shock protein,
mitochondrial precursor
IPI00219005
15
FKBP4 FK506-binding
10
2
8
0
protein 4
IPI00003519
13
EFTUD2 116 kDa U5
10
1
8
0
small nuclear
ribonucleoprotein
component
IPI00013774
4
HDAC1 Histone
10
3
8
0
deacetylase 1
IPI00428967
6
TICAM2; TMED7 Toll-
10
1
8
0
like receptor adapter
molecule 2
IPI00219445
5
PSME3 Isoform 2 of
10
1
8
0
Proteasome activator
complex subunit 3
IPI00029731
4
RPL35A 60S ribosomal
10
2
8
0
protein L35a
IPI00025329
3
RPL19 60S ribosomal
10
4
8
0
protein L19
IPI00455757
3
Similar to 60S
10
2
8
0
ribosomal protein L35
IPI00009407
2
DAD1 Dolichyl-
10
0
8
0
diphosphooligosaccharide--
protein
glycosyltransferase
subunit DAD1
IPI00010951
33
EPPK1 Epiplakin
9
1
8
0
IPI00658109
16
LOC100133623; CKMT1B;
9
9
8
0
CKMT1A Creatine
kinase, ubiquitous
mitochondrial precursor
IPI00293721
9
AKR7A3 Aflatoxin B1
9
4
8
0
aldehyde reductase
member 3
IPI00642211
15
RNPEP Aminopeptidase B
9
2
8
0
IPI00299608
14
PSMD1 Isoform 1 of
9
2
8
0
26S proteasome non-
ATPase regulatory
subunit 1
IPI00017726
13
HSD17B10 Isoform 1 of
9
2
8
0
3-hydroxyacyl-CoA
dehydrogenase type-2
IPI00479722
13
PSME1 Proteasome
9
1
8
0
activator complex
subunit 1
IPI00009750
12
LGALS4 Galectin-4
9
6
8
0
IPI00334190
10
STOML2 Stomatin-like
9
2
8
0
protein 2
IPI00853220
10
SEC31A Isoform 6 of
9
2
8
0
Protein transport protein
Sec31A
IPI00030131
9
TMPO Isoform Beta of
9
2
8
0
Lamina-associated
polypeptide 2, isoforms
beta/gamma
IPI00640155
8
PSMB8 proteasome beta
9
3
8
0
8 subunit isoform E2
proprotein
IPI00221222
7
SUB1 Activated RNA
9
2
8
0
polymerase II
transcriptional
coactivator p15
IPI00465361
6
RPL13 60S ribosomal
9
2
8
0
protein L13
IPI00005537
6
MRPL12 39S ribosomal
9
4
8
1
protein L12,
mitochondrial precursor
IPI00293564
5
HMGCL
9
3
8
0
Hydroxymethylglutaryl-
CoA lyase,
mitochondrial precursor
IPI00785096
4
BZW1 Isoform 1 of
9
1
8
0
Basic leucine zipper and
W2 domain-containing
protein 1
IPI00100656
4
GPSN2 Isoform 1 of
9
0
8
1
Synaptic glycoprotein
SC2
IPI00009922
4
C14orf156 SRA stem-
9
1
8
0
loop-interacting RNA-
binding protein,
mitochondrial precursor
IPI00007188
17
SLC25A5 ADP/ATP
8
2
8
0
translocase 2
IPI00017375
9
SEC23A Protein
8
1
8
0
transport protein
Sec23A
IPI00873762
3
TAF15 65 kDa protein
8
0
8
0
IPI00885213
7
TXNRD1 Isoform 6 of
8
1
8
3
Thioredoxin reductase 1,
cytoplasmic
IPI00329352
6
NOMO1; NOMO3
8
3
8
0
Nodal modulator 1
precursor
IPI00029628
3
RCN2 Reticulocalbin-2
8
0
8
0
precursor
IPI00797230
2
RPL8 32 kDa protein
8
3
8
0
IPI00220835
2
SEC61B Protein
8
2
8
0
transport protein Sec61
subunit beta
IPI00784366
13
AP2B1 Isoform 2 of
7
0
8
0
AP-2 complex subunit
beta-1
IPI00220994
5
H2AFY2 Core histone
7
1
8
0
macro-H2A.2
IPI00012268
12
PSMD2 26S proteasome
7
2
8
0
non-ATPase regulatory
subunit 2
IPI00013068
12
EIF3E Eukaryotic
7
0
8
0
translation initiation
factor 3 subunit E
IPI00472054
11
FAM120A Isoform A of
7
4
8
0
Constitutive coactivator
of PPAR-gamma-like
protein 1
IPI00383046
6
CMBL
7
8
8
1
Carboxymethylenebutenolidase
homolog
IPI00031820
5
FARSA Phenylalanyl-
7
2
8
0
tRNA synthetase alpha
chain
IPI00004845
4
NIPSNAP3A Protein
7
3
8
0
NipSnap homolog 3A
IPI00018465
24
CCT7 T-complex
6
2
8
0
protein 1 subunit eta
IPI00027442
19
AARS Alanyl-tRNA
6
3
8
0
synthetase, cytoplasmic
IPI00017592
9
LETM1 Leucine zipper-
6
2
8
0
EF-hand-containing
transmembrane protein
1, mitochondrial
precursor
IPI00300050
8
HSD11B2
6
13
8
2
Corticosteroid 11-beta-
dehydrogenase isozyme 2
IPI00306516
8
TIMM44 Import inner
6
1
8
0
membrane translocase
subunit TIM44,
mitochondrial precursor
IPI00293267
5
LGALS9 Isoform Short
6
3
8
0
of Galectin-9
IPI00021258
7
ARFIP1 Isoform B of
6
2
8
1
Arfaptin-1
IPI00303568
5
PTGES2 Prostaglandin
6
1
8
0
E synthase 2
IPI00003870
5
CLPP Putative ATP-
6
2
8
0
dependent Clp protease
proteolytic subunit,
mitochondrial precursor
IPI00843910
4
FUCA1 Tissue alpha-L-
6
16
8
4
fucosidase precursor
IPI00452747
4
LOC653566 Similar to
6
1
8
0
Signal peptidase
complex subunit 2
IPI00304612
2
RPL13A 60S ribosomal
6
1
8
0
protein L13a
IPI00871366
9
RAB1B Small GTP-
5
1
8
0
binding protein
IPI00006451
9
NSF Vesicle-fusing
5
0
8
1
ATPase
IPI00025239
6
NDUFS2 NADH
5
2
8
0
dehydrogenase
[ubiquinone] iron-sulfur
protein 2, mitochondrial
precursor
IPI00550644
5
LL22NC03-5H6.5
5
3
8
0
UPF0530 protein
IPI00016077
3
GBAS Protein NipSnap
5
6
8
0
homolog 2
IPI00031534
2
ST6GALNAC1 Alpha-
5
5
8
0
N-acetylgalactosaminide
alpha-2,6-
sialyltransferase 1
IPI00025796
9
NDUFS3 NADH
4
1
8
0
dehydrogenase
[ubiquinone] iron-sulfur
protein 3, mitochondrial
precursor
IPI00794899
3
37 kDa protein
4
4
8
0
IPI00029054
4
NT5C2 Cytosolic purine
4
0
8
0
5′-nucleotidase
IPI00002255
4
LRBA
4
3
8
0
Lipopolysaccharide-
responsive and beige-
like anchor protein
IPI00253050
2
L1TD1 LINE-1 type
4
0
8
0
transposase domain-
containing protein 1
IPI00465179
8
PFKM cDNA FLJ44241
3
1
8
0
fis, clone
THYMU3008436,
highly similar to 6-
phosphofructokinase,
muscle type
IPI00294187
9
PADI2 Protein-arginine
3
13
8
2
deiminase type-2
IPI00102581
7
SULT1B1
3
9
8
0
Sulfotransferase family
cytosolic 1B member 1
IPI00023647
4
UBA6 Isoform 1 of
3
2
8
0
Ubiquitin-like modifier-
activating enzyme 6
IPI00030320
5
DDX6 Probable ATP-
2
1
8
0
dependent RNA helicase
DDX6
IPI00220740
8
NPM1 Isoform 2 of
17
2
7
0
Nucleophosmin
IPI00386755
7
ERO1L ERO1-like
17
1
7
1
protein alpha precursor
IPI00295542
13
NUCB1 Nucleobindin-1
15
3
7
0
precursor
IPI00293655
13
DDX1 ATP-dependent
14
1
7
0
RNA helicase DDX1
IPI00007084
11
SLC25A13
14
2
7
0
Mitochondrial aspartate-
glutamate carrier protein
IPI00007427
8
AGR2 AGR2
14
5
7
0
IPI00152409
7
AGR3 Anterior gradient
14
4
7
0
protein 3 homolog
precursor
IPI00004573
27
PIGR Polymeric
13
5
7
1
immunoglobulin
receptor precursor
IPI00021290
15
ACLY ATP-citrate
13
1
7
0
synthase
IPI00456919
14
HUWE1 Isoform 1 of
13
0
7
0
E3 ubiquitin-protein
ligase HUWE1
IPI00015018
13
PPA1 Inorganic
13
2
7
0
pyrophosphatase
IPI00218493
8
HPRT1 Hypoxanthine-
13
1
7
0
guanine
phosphoribosyltransferase
IPI00876962
6
INF2 Isoform 2 of
13
2
7
0
Inverted formin-2
IPI00479997
5
STMN1 Stathmin
13
1
7
0
IPI00791426
5
RPL24 13 kDa protein
13
1
7
0
IPI00215719
4
RPL18 60S ribosomal
13
1
7
0
protein L18
IPI00334713
9
HNRNPAB Isoform 3
12
3
7
0
of Heterogeneous
nuclear
ribonucleoprotein A/B
IPI00012442
8
G3BP1 Ras GTPase-
12
0
7
0
activating protein-
binding protein 1
IPI00301311
8
SET Isoform 2 of
12
2
7
0
Protein SET
IPI00011913
6
HNRNPA0
12
2
7
0
Heterogeneous nuclear
ribonucleoprotein A0
IPI00413673
5
BCLAF1 Isoform 4 of
12
3
7
0
Bcl-2-associated
transcription factor 1
IPI00059292
4
MAGOHB Protein
12
0
7
1
mago nashi homolog 2
IPI00409635
3
FAM62B Isoform 2 of
12
0
7
0
Extended
synaptotagmin-2
IPI00555747
9
PABPC4 Isoform 2 of
11
2
7
0
Polyadenylate-binding
protein 4
IPI00604620
25
NCL Isoform 1 of
11
3
7
0
Nucleolin
IPI00000877
24
HYOU1 Hypoxia up-
11
2
7
0
regulated protein 1
precursor
IPI00141318
24
CKAP4 Isoform 1 of
11
2
7
0
Cytoskeleton-associated
protein 4
IPI00217468
10
HIST1H1B Histone
11
2
7
0
H1.5
IPI00797148
15
HNRNPA1 HNRPA1
11
2
7
0
protein
IPI00438229
15
TRIM28 Isoform 1 of
11
1
7
0
Transcription
intermediary factor 1-
beta
IPI00031812
6
YBX1 Nuclease-
11
2
7
0
sensitive element-
binding protein 1
IPI00221092
8
RPS16 40S ribosomal
11
2
7
0
protein S16
IPI00396321
8
LRRC59 Leucine-rich
11
3
7
0
repeat-containing
protein 59
IPI00009659
4
C20orf77
11
1
7
0
Uncharacterized protein
C20orf77
IPI00157790
4
KIAA0368 KIAA0368
11
0
7
0
protein
IPI00794978
2
MRPL47 MRPL47
11
1
7
0
protein
IPI00004524
2
GCA Grancalcin
11
0
7
0
IPI00217437
7
TTBK2 Tau-tubulin
10
5
7
0
kinase
IPI00880104
3
KRT74 59 kDa protein
10
3
7
0
IPI00550661
3
KRT13 Isoform 2 of
10
5
7
0
Keratin, type I
cytoskeletal 13
IPI00382470
44
HSP90AA1 heat shock
10
1
7
0
protein 90 kDa alpha
(cytosolic), class A
member 1 isoform 1
IPI00651677
24
DDX17 Isoform 2 of
10
2
7
0
Probable ATP-
dependent RNA helicase
DDX17
IPI00017617
20
DDX5 Probable ATP-
10
3
7
0
dependent RNA helicase
DDX5
IPI00010471
29
LCP1 Plastin-2
10
1
7
1
IPI00007765
34
HSPA9 Stress-70
10
2
7
0
protein, mitochondrial
precursor
IPI00298520
16
ARCN1 Putative
10
2
7
0
uncharacterized protein
DKFZp686M09245
IPI00217030
14
RPS4X 40S ribosomal
10
2
7
0
protein S4, X isoform
IPI00000494
13
RPL5 60S ribosomal
10
2
7
0
protein L5
IPI00012585
10
HEXB Beta-
10
3
7
0
hexosaminidase subunit
beta precursor
IPI00026105
10
SCP2 Isoform SCPx of
10
4
7
0
Non-specific lipid-
transfer protein
IPI00008455
8
MYO6 Isoform 2 of
10
1
7
0
Myosin-VI
IPI00399183
7
APOB48R Isoform 1 of
10
5
7
1
Apolipoprotein B-100
receptor
IPI00744364
6
SFRS7 Uncharacterized
10
4
7
0
protein SFRS7
IPI00465132
5
COPE Coatomer subunit
10
1
7
0
epsilon
IPI00607584
5
MYBBP1A Isoform 2 of
10
0
7
0
Myb-binding protein 1A
IPI00787692
4
LOC650788 similar to
10
2
7
0
40S ribosomal protein
S28
IPI00014808
3
PAFAH1B3 Platelet-
10
0
7
0
activating factor
acetylhydrolase IB
subunit gamma
IPI00019449
3
LOC100133484; HLA-
10
9
7
1
DQB1; HLA-
DRB1; hCG_1998957; HLA-
DRB4; LOC100133811;
LOC100133583; HLA-
DRB3; HLA-
DRB5; HLA-
DQB2; HLA-
DRB2; ZNF749; LOC100133661;
RNASE2 Non-
secretory ribonuclease
precursor
IPI00217975
31
LMNB1 Lamin-B1
9
3
7
1
IPI00455599
12
HSP90AB2P Heat shock
9
2
7
0
protein 90Bb
IPI00853059
14
FUBP1 Isoform 2 of Far
9
1
7
0
upstream element-
binding protein 1
IPI00218852
30
VIL1 Villin-1
9
3
7
0
IPI00789551
15
MATR3
9
1
7
0
Uncharacterized protein
MATR3
IPI00410693
11
SERBP1 Isoform 1 of
9
4
7
0
Plasminogen activator
inhibitor 1 RNA-binding
protein
IPI00646899
10
RPL10 Ribosomal
9
2
7
0
protein L10
IPI00220362
9
HSPE1 10 kDa heat
9
3
7
0
shock protein,
mitochondrial
IPI00299573
9
RPL7A 60S ribosomal
9
2
7
0
protein L7a
IPI00221354
5
FUS Isoform Short of
9
2
7
0
RNA-binding protein
FUS
IPI00893715
7
TACSTD1 38 kDa
9
4
7
0
protein
IPI00872533
6
CD2AP 76 kDa protein
9
1
7
0
IPI00006181
6
EIF3D Eukaryotic
9
3
7
0
translation initiation
factor 3 subunit D
IPI00009368
5
SFXN1 Sideroflexin-1
9
2
7
1
IPI00219516
4
GUSB Isoform Short of
9
2
7
0
Beta-glucuronidase
precursor
IPI00398135
4
hCG_21078
9
2
7
0
hypothetical protein
LOC389435
IPI00082310
3
KHDRBS1 Isoform 3 of
9
1
7
0
KH domain-containing,
RNA-binding, signal
transduction-associated
protein 1
IPI00328268
2
EIF4G3 EIF4G3 protein
9
0
7
0
IPI00644079
21
HNRNPU
8
2
7
0
heterogeneous nuclear
ribonucleoprotein U
isoform a
IPI00414980
9
MYO1B Isoform 2 of
8
5
7
4
Myosin-Ib
IPI00646304
13
PPIB peptidylprolyl
8
3
7
0
isomerase B precursor
IPI00017334
13
PHB Prohibitin
8
1
7
0
IPI00744692
13
TALDO1 Transaldolase
8
1
7
0
IPI00329633
12
TARS Threonyl-tRNA
8
1
7
0
synthetase, cytoplasmic
IPI00030009
11
PAPSS2 Isoform A of
8
10
7
1
Bifunctional 3′-
phosphoadenosine 5′-
phosphosulfate
synthetase 2
IPI00027851
7
HEXA Beta-
8
3
7
0
hexosaminidase subunit
alpha precursor
IPI00013174
7
RBM14 Isoform 1 of
8
3
7
0
RNA-binding protein 14
IPI00024933
7
RPL12 60S ribosomal
8
2
7
0
protein L12
IPI00793375
6
XPNPEP1 Xaa-Pro
8
1
7
0
aminopeptidase 1
IPI00219160
5
RPL34 60S ribosomal
8
4
7
0
protein L34
IPI00010949
5
SIAE Isoform 1 of
8
7
7
1
Sialate O-acetylesterase
precursor
IPI00005737
4
SURF4 Isoform 1 of
8
1
7
0
Surfeit locus protein 4
IPI00029631
3
ERH Enhancer of
8
0
7
0
rudimentary homolog
IPI00029601
15
CTTN Src substrate
7
3
7
0
cortactin
IPI00030179
11
RPL7 60S ribosomal
7
3
7
0
protein L7
IPI00644570
3
18 kDa protein
7
4
7
0
IPI00456887
13
HNRNPUL2
7
0
7
0
Heterogeneous nuclear
ribonucleoprotein U-like
protein 2
IPI00031169
11
RAB2A Ras-related
7
0
7
0
protein Rab-2A
IPI00016910
11
EIF3CL; EIF3C
7
3
7
0
Eukaryotic translation
initiation factor 3
subunit C
IPI00031583
11
USO1 Putative
7
4
7
4
uncharacterized protein
DKFZp451D234
IPI00165360
9
MPST 3-
7
3
7
0
mercaptopyruvate
sulfurtransferase
IPI00025019
7
PSMB1 Proteasome
7
2
7
0
subunit beta type-1
precursor
IPI00335930
4
DAZAP1 Isoform 2 of
7
1
7
0
DAZ-associated protein 1
IPI00015029
4
PTGES3 Prostaglandin
7
2
7
0
E synthase 3
IPI00029039
4
REG3A Regenerating
7
0
7
0
islet-derived protein 3
alpha precursor
IPI00061525
3
GNPNAT1
7
0
7
0
Glucosamine 6-
phosphate N-
acetyltransferase
IPI00026167
3
NHP2L1 NHP2-like
7
0
7
0
protein 1
IPI00000811
3
PSMB6 Proteasome
7
2
7
1
subunit beta type-6
precursor
IPI00790799
3
SEC11A 16 kDa protein
7
0
7
0
IPI00413654
3
SFRS5 Isoform SRP40-
7
2
7
0
4 of Splicing factor,
arginine/serine-rich 5
IPI00006092
3
PMM2
7
1
7
0
Phosphomannomutase 2
IPI00328840
3
THOC4 THO complex
7
1
7
0
subunit 4
IPI00216237
3
RPL36 60S ribosomal
7
3
7
1
protein L36
IPI00173589
2
LOC284064 similar to
7
3
7
0
ribosomal protein L29
IPI00009342
64
IQGAP1 Ras GTPase-
6
2
7
0
activating-like protein
IQGAP1
IPI00413947
10
AP1B1 Isoform B of
6
0
7
0
AP-1 complex subunit
beta-1
IPI00398798
5
H2AFV H2A histone
6
4
7
0
family, member V
isoform 3
IPI00303882
13
M6PRBP1 Isoform B of
6
2
7
0
Mannose-6-phosphate
receptor-binding protein 1
IPI00790743
5
Protein
6
5
7
0
IPI00021800
8
CASP1 Isoform Alpha
6
5
7
0
of Caspase-1 precursor
IPI00060181
7
EFHD2 EF-hand
6
0
7
1
domain-containing
protein D2
IPI00017510
6
MT-CO2 Cytochrome c
6
6
7
0
oxidase subunit 2
IPI00006443
6
CRYL1 Lambda-
6
6
7
0
crystallin homolog
IPI00020956
6
HDGF Hepatoma-
6
2
7
0
derived growth factor
IPI00060200
5
GALM Aldose 1-
6
6
7
0
epimerase
IPI00185374
5
PSMD12 26S
6
1
7
0
proteasome non-ATPase
regulatory subunit 12
IPI00012340
4
SFRS9 Splicing factor,
6
3
7
1
arginine/serine-rich 9
IPI00016405
4
OCIAD1 Isoform 1 of
6
5
7
0
OCIA domain-
containing protein 1
IPI00019329
3
DYNLL1 Dynein light
6
4
7
0
chain 1, cytoplasmic
IPI00642816
3
SRP9; hCG_1781062
6
2
7
0
Signal recognition
particle 9 kDa protein
IPI00013968
3
COX7C Cytochrome c
6
2
7
0
oxidase subunit 7C,
mitochondrial precursor
IPI00026570
2
COX7A2 Cytochrome c
6
4
7
0
oxidase polypeptide
VIIa-liver/heart,
mitochondrial precursor
IPI00005719
10
RAB1A Isoform 1 of
5
2
7
0
Ras-related protein Rab-
1A
IPI00374686
6
Uncharacterized protein
5
1
7
0
ENSP00000341227
(Fragment)
IPI00219077
15
LTA4H Isoform 1 of
5
2
7
0
Leukotriene A-4
hydrolase
IPI00025366
13
CS Citrate synthase,
5
1
7
1
mitochondrial precursor
IPI00290110
13
PDCD4 Programmed
5
5
7
0
cell death protein 4
IPI00332828
10
CES2 carboxylesterase 2
5
7
7
5
isoform 1
IPI00182757
7
KIAA1967 Isoform 1 of
5
3
7
0
Protein KIAA1967
IPI00030654
6
CPSF6 Isoform 2 of
5
1
7
0
Cleavage and
polyadenylation
specificity factor subunit 6
IPI00789848
5
IVD Isovaleryl-CoA
5
7
7
0
dehydrogenase,
mitochondrial precursor
IPI00024934
4
MUT Methylmalonyl-
5
0
7
0
CoA mutase,
mitochondrial precursor
IPI00015972
4
COX6C Cytochrome c
5
6
7
0
oxidase polypeptide VIc
precursor
IPI00031772
3
C15orf48 Normal
5
7
7
0
mucosa of esophagus-
specific gene 1 protein
IPI00019888
2
ALDH5A1 Succinate-
5
0
7
0
semialdehyde
dehydrogenase,
mitochondrial precursor
IPI00873259
2
ATP5J2 10 kDa protein
5
2
7
0
IPI00005159
11
ACTR2 Actin-related
4
1
7
0
protein 2
IPI00465256
8
AK3 GTP:AMP
4
2
7
1
phosphotransferase
mitochondrial
IPI00513827
8
ACADM Putative
4
4
7
0
uncharacterized protein
DKFZp686M24262
IPI00554811
6
ARPC4; TTLL3 Actin-
4
2
7
0
related protein 2/3
complex subunit 4
IPI00874156
6
OTUB1 Isoform 1 of
4
2
7
0
Ubiquitin thioesterase
OTUB1
IPI00024661
4
SEC24C Protein
4
2
7
1
transport protein Sec24C
IPI00028387
4
C20orf116 Isoform 1 of
4
1
7
0
Uncharacterized protein
C20orf116 precursor
IPI00022277
4
CCDC56 Coiled-coil
4
6
7
0
domain-containing
protein 56
IPI00024742
2
UQCRQ Cytochrome b-
4
3
7
0
c1 complex subunit 8
IPI00307547
2
C9orf46
4
1
7
0
Uncharacterized protein
C9orf46
IPI00029133
10
ATP5F1 ATP synthase
3
3
7
0
subunit b, mitochondrial
precursor
IPI00651719
6
PBLD MAWD binding
3
2
7
1
protein isoform b
IPI00219755
1
SPCS1 Signal peptidase
3
3
7
0
complex subunit 1
IPI00880101
9
CEACAM5 Protein
29
0
6
0
IPI00302944
51
COL12A1 Isoform 4 of
24
3
6
0
Collagen alpha-1(XII)
chain precursor
IPI00027769
5
ELA2 Leukocyte
23
0
6
0
elastase precursor
IPI00026781
46
FASN Fatty acid
20
2
6
0
synthase
IPI00304754
5
FERMT1 Isoform 1 of
18
3
6
0
Fermitin family
homolog 1
IPI00019472
5
SLC1A5 Neutral amino
16
0
6
0
acid transporter B
IPI00026833
5
ADSS Adenylosuccinate
16
0
6
0
synthetase isozyme 2
IPI00026328
3
TXNDC12 Thioredoxin
14
2
6
0
domain-containing
protein 12 precursor
IPI00009904
36
PDIA4 Protein
13
3
6
0
disulfide-isomerase A4
precursor
IPI00216044
8
RALY Isoform 1 of
13
0
6
0
RNA-binding protein
Raly
IPI00215790
6
RPL38 60S ribosomal
13
2
6
0
protein L38
IPI00872940
6
RPL30 Uncharacterized
13
1
6
0
protein RPL30
(Fragment)
IPI00012007
11
AHCY
12
2
6
0
Adenosylhomocysteinase
IPI00012069
6
NQO1 NAD
12
1
6
0
IPI00385267
5
SRPR Signal
12
1
6
0
recognition particle
receptor subunit alpha
IPI00856058
4
RPL31 ribosomal
12
3
6
0
protein L31 isoform 3
IPI00395865
3
RBBP7 Histone-binding
12
0
6
0
protein RBBP7
IPI00215734
3
PRMT1 Isoform 2 of
12
0
6
0
Protein arginine N-
methyltransferase 1
IPI00554648
51
KRT8 Keratin, type II
11
3
6
0
cytoskeletal 8
IPI00027201
64
MUC2 Mucin-2
11
4
6
1
precursor
IPI00789401
22
PLS1 PLS1 protein
11
2
6
0
IPI00215743
48
RRBP1 Isoform 3 of
11
3
6
0
Ribosome-binding
protein 1
IPI00003881
11
HNRNPF
11
2
6
0
Heterogeneous nuclear
ribonucleoprotein F
IPI00410017
18
PABPC1 Isoform 2 of
11
2
6
0
Polyadenylate-binding
protein 1
IPI00022228
21
HDLBP Vigilin
11
2
6
0
IPI00401264
11
TXNDC4 Thioredoxin
11
2
6
0
domain-containing
protein 4 precursor
IPI00008438
9
RPS10 40S ribosomal
11
2
6
0
protein S10
IPI00295741
9
CTSB Cathepsin B
11
5
6
0
precursor
IPI00004968
7
PRPF19 Pre-mRNA-
11
0
6
1
processing factor 19
IPI00014230
6
C1QBP Complement
11
2
6
0
component 1 Q
subcomponent-binding
protein, mitochondrial
precursor
IPI00646917
4
NUDT21 Cleavage and
11
0
6
0
polyadenylation
specificity factor subunit 5
IPI00219155
4
RPL27 60S ribosomal
11
1
6
0
protein L27
IPI00218606
4
RPS23 40S ribosomal
11
5
6
0
protein S23
IPI00101405
4
FDPS Farnesyl
11
2
6
0
diphosphate synthase
IPI00028006
3
PSMB2 Proteasome
11
2
6
0
subunit beta type-2
IPI00386662
2
FUSIP1 Isoform 4 of
11
1
6
0
FUS-interacting serine-
arginine-rich protein 1
IPI00027230
43
HSP90B1 Endoplasmin
10
2
6
0
precursor
IPI00027834
15
HNRNPL
10
1
6
0
heterogeneous nuclear
ribonucleoprotein L
isoform a
IPI00219870
23
CTNND1 Isoform 1A of
10
3
6
0
Catenin delta-1
IPI00383296
21
HNRNPM Isoform 2 of
10
2
6
0
Heterogeneous nuclear
ribonucleoprotein M
IPI00020599
19
CALR Calreticulin
10
2
6
0
precursor
IPI00644989
18
PDIA6 Isoform 1 of
10
2
6
0
Protein disulfide-
isomerase A6 precursor
IPI00646486
9
HP1BP3
10
2
6
0
Heterochromatin protein
1, binding protein 3
IPI00010896
15
CLIC1 Chloride
10
2
6
0
intracellular channel
protein 1
IPI00000690
13
AIFM1 Isoform 1 of
10
4
6
0
Apoptosis-inducing
factor 1, mitochondrial
precursor
nPeptides, number of peptides that were identified in all the samples analyzed;
nBigT, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was >3;
nSmallT, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was <1/3;
nBigP, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was >3;
nSmallP, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was <1/3.
Table 4 lists proteins that were observed to be highly expressed in polyps, whereas in more advanced stages of colorectal cancer these proteins tended to have decreased levels of expression. Accordingly, a diagnostic array of reagents directed to detection of at least some of the proteins in this group may be used as a screening test for very early detection of colorectal cancer. Such a screening test could identify susceptible at-risk individuals, even prior to the stage at which polyp visualization is possible by endoscopic techniques.
TABLE 4
Protein
(IPI Acc. No.)
nBigTumor
nSmallTumor
nBigPolyp
nSmallPolyp
CPT2 Carnitine O-
6
5
12
0
palmitoyltransferase 2,
mitochondrial precursor
(IPI00012912)
ARL1 ADP-ribosylation factor-
6
1
12
0
like protein 1
(IPI00219518)
PFKL Isoform 1 of 6-
6
2
11
0
phosphofructokinase, liver type
(IPI00332371)
GOT2 Aspartate
6
3
11
0
aminotransferase, mitochondrial
precursor
(IPI00018206)
AP1G1 AP-1 complex subunit
6
1
11
0
gamma-1
(IPI00643591)
STRBP Isoform 2 of Spermatid
6
0
11
0
perinuclear RNA-binding protein
(IPI00413860)
CLCA1 Calcium-activated
4
13
11
1
chloride channel regulator 1
precursor
(IPI00014625)
CYFIP1 Isoform 1 of
4
2
11
0
Cytoplasmic FMR1-interacting
protein 1
(IPI00644231)
COQ9 Isoform 1 of Ubiquinone
4
3
11
0
biosynthesis protein COQ9,
mitochondrial precursor
(IPI00470631)
NDUFA9 NADH dehydrogenase
3
4
11
0
[ubiquinone] 1 alpha subcomplex
subunit 9, mitochondrial
precursor
(IPI00003968)
ALDH7A1 Similar to Antiquitin
2
5
11
0
(IPI00221234)
HMGCS1
6
4
10
0
Hydroxymethylglutaryl-CoA
synthase, cytoplasmic
(IPI00008475)
NNT NAD(P) transhydrogenase,
6
2
10
0
mitochondrial precursor
(IPI00337541)
PRDX5 Uncharacterized protein
6
2
10
0
PRDX5 (Fragment)
(IPI00876999)
PCCB Propionyl-CoA
6
5
10
0
carboxylase beta chain,
mitochondrial precursor
(IPI00007247)
COPZ1 Coatomer subunit zeta-1
6
4
10
0
(IPI00032851)
BAX BCL2-associated X protein
6
2
10
0
isoform sigma
(IPI00845474)
ACAD9 Acyl-CoA
6
0
10
0
dehydrogenase family member 9,
mitochondrial precursor
(IPI00152981)
UBXD8 UBX domain-containing
6
1
10
0
protein 8
(IPI00172656)
HMGCS2
5
12
10
0
Hydroxymethylglutaryl-CoA
synthase, mitochondrial precursor
(IPI00008934)
SLC25A3 cDNA FLJ90278 fis,
5
1
10
0
clone NT2RP1000325, highly
similar to Phosphate carrier
protein, mitochondrialprecursor
(IPI00790115)
SLC25A11 Mitochondrial 2-
5
4
10
0
oxoglutarate/malate carrier
protein
(IPI00219729)
PDCD6 Programmed cell death
5
0
10
0
protein 6
(IPI00025277)
UCRC Cytochrome b-c1 complex
4
0
10
0
subunit 9
(IPI00554701)
DEFA6 Defensin-6 precursor
4
0
10
0
(IPI00008301)
DYNC1H1 Cytoplasmic dynein 1
3
1
10
0
heavy chain 1
(IPI00456969)
HK1 Isoform 2 of Hexokinase-1
3
1
10
0
(IPI00220663)
CYFIP2 Isoform 2 of
3
2
10
0
Cytoplasmic FMR1-interacting
protein 2
(IPI00719600)
DCI Isoform 2 of 3,2-trans-enoyl-
3
5
10
0
CoA isomerase, mitochondrial
precursor
(IPI00398758)
CISD1 CDGSH iron sulfur
1
1
10
0
domain-containing protein 1
(IPI00020510)
nBigTumor, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was >3;
nSmallTumor, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was <1/3;
nBigPolyp, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was >3;
nSmallPolyp, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was <1/3.
The proteins identified by isotopic labeling included those listed in Table 5. All of the listed proteins were found to be present in colon cancer tissue at levels that were at least 2.5-fold greater than the level of the same protein in healthy colon tissue from the same subject, as indicated by the median ratios of the protein levels (i.e. cancerous tissue:healthy tissue) listed in the column denoted “MED”.
TABLE 5
Protein Name
IPI Acc. No.
nPEP
MED
#>2.5
DPEP1 Dipeptidase 1 precursor
IPI00059476
12
50
8
LCN2 Lipocalin 2
IPI00643623
6
48.75
13
FAM62B Isoform 2 of Extended
IPI00409635
10
39.73
10
synaptotagmin-2
MTA2 Metastasis-associated
IPI00171798
8
31.45
8
protein MTA2
S100A8 Protein S100-A8
IPI00007047
7
30.72
17
S100A9 Protein S100-A9
IPI00027462
7
28.205
17
MPO Isoform H7 of
IPI00236556
26
25.57
14
Myeloperoxidase precursor
MCM2 DNA replication licensing
IPI00184330
12
23.37
11
factor MCM2
FDFT1 Squalene synthetase
IPI00020944
5
23.155
7
DMBT1 Isoform 1 of Deleted in
IPI00099110
7
22.86
7
malignant brain tumors 1 protein
precursor
LTF Lactotransferrin precursor
IPI00848342
30
20.45
17
SERPINB5 Serpin B5 precursor
IPI00783625
7
20.44
8
OLFM4 Olfactomedin-4 precursor
IPI00022255
20
19.49
13
FERMT1 Isoform 1 of Fermitin
IPI00304754
9
16.05
14
family homolog 1
C1R; ACYP1; C17orf13
IPI00296165
5
15.61
7
Complement C1r
subcomponent precursor
PLOD3 Procollagen-lysine, 2-
IPI00030255
10
15.17
8
oxoglutarate 5-dioxygenase 3
precursor
CEACAM5 Carcinoembryonic
IPI00027486
9
14.12
17
antigen-related cell adhesion
molecule 5 precursor
PYCR1 pyrroline-5-carboxylate
IPI00376503
6
12.505
12
reductase 1 isoform 2
MCM6 DNA replication licensing
IPI00031517
9
12.29
6
factor MCM6
SLC1A5 Neutral amino acid
IPI00019472
8
10.03
10
transporter B
THBS1 Thrombospondin-1
IPI00296099
13
8.55
14
precursor
LACTB2 Beta-lactamase-like
IPI00006952
5
8.445
8
protein 2
NAT10 N-acetyltransferase 10
IPI00300127
11
7.85
10
RSL1D1 RSL1D1 protein
IPI00642046
11
7.845
12
LMO7 Isoform 3 of LIM domain
IPI00291802
11
7.74
10
only protein 7
LYZ Lysozyme C precursor
IPI00019038
7
7.64
15
MCM7 Isoform 1 of DNA
IPI00299904
10
7.435
7
replication licensing
factor MCM7
F11R Junctional adhesion molecule
IPI00001754
5
6.775
7
A precursor
MCM3 DNA replication licensing
IPI00013214
11
6.605
10
factor MCM3
ATP6V1E1 vacuolar H+ ATPase
IPI00719806
5
6.34
9
E1 isoform b
TNC Isoform 1 of Tenascin
IPI00031008
58
6.2
12
precursor
COL12A1 Isoform 1 of Collagen
IPI00329573
49
6.175
17
alpha-1(XII) chain precursor
SORD 11 kDa protein
IPI00791243
5
5.78
7
PYCR2 Pyrroline-5-carboxylate
IPI00470610
5
5.645
9
reductase 2
GTF2I Isoform 2 of General
IPI00293242
10
5.515
7
transcription factor II-I
DDX18 ATP-dependent RNA
IPI00301323
8
5.38
8
helicase DDX18
RBM39 Isoform 2 of RNA-binding
IPI00215801
11
5.19
12
protein 39
NQO1 NAD
IPI00012069
6
4.9
8
DNAJA3 Isoform 2 of DnaJ
IPI00179187
7
4.73
9
homolog subfamily A
member 3, mitochondrial
precursor
NCBP1 Nuclear cap-binding
IPI00019380
8
4.42
9
protein subunit 1
HSPH1 Isoform Beta of Heat
IPI00218993
33
4.365
13
shock protein 105 kDa
ADSS Adenylosuccinate
IPI00026833
7
4.36
11
synthetase isozyme 2
PSAT1 Isoform 1 of Phosphoserine
IPI00001734
9
4.295
8
aminotransferase
ALG5 Dolichyl-phosphate beta-
IPI00002506
6
4.135
10
glucosyltransferase
PCNA Proliferating cell nuclear
IPI00021700
12
4.04
10
antigen
TCOF1 Isoform 2 of Treacle
IPI00298696
12
3.92
9
protein
SERPINH1 Serpin H1 precursor
IPI00032140
20
3.9
16
ERO1L ERO1-like protein alpha
IPI00386755
8
3.81
12
precursor
ILVBL Isoform 1 of Acetolactate
IPI00554541
15
3.8
11
synthase-like protein
ANXA3 Annexin A3
IPI00024095
17
3.645
16
NAMPT Isoform 1 of
IPI00018873
25
3.61
12
Nicotinamide
phosphoribosyltransferase
TFRC Transferrin receptor
IPI00022462
23
3.56
9
protein 1
SERPINB9 Serpin B9
IPI00032139
8
3.41
10
EIF2S2 Eukaryotic translation
IPI00021728
8
3.38
8
initiation factor 2 subunit 2
SRRM2 Isoform 1 of Serine/
IPI00782992
8
3.365
8
arginine repetitive
matrix protein 2
ARHGEF1 Isoform 1 of Rho
IPI00647786
5
3.31
7
guanine nucleotide
exchange factor 1
COMT Isoform Soluble of
IPI00375513
9
3.31
10
Catechol O-methyltransferase
DEK 48 kDa protein
IPI00871695
8
3.285
10
SYK Isoform Long of Tyrosine-
IPI00018597
6
3.275
7
protein kinase SYK
S100A11 Protein S100-A11
IPI00013895
8
3.2
12
HSDL2 Isoform 1 of
IPI00414384
10
3.14
10
Hydroxysteroid
dehydrogenase-like protein 2
C7orf24 Uncharacterized protein
IPI00031564
5
3.125
9
C7orf24
HM13 Isoform 1 of Minor
IPI00152441
7
3.09
9
histocompatibility antigen H13
RCN1 Reticulocalbin-1 precursor
IPI00015842
14
3.07
11
DIAPH1 Diaphanous homolog 1
IPI00884341
10
3.06
8
SRM Spermidine synthase
IPI00292020
5
2.97
11
ATAD3A Isoform 2 of ATPase
IPI00295992
20
2.965
10
family AAA domain-containing
protein 3A
GPX2 Glutathione peroxidase 2
IPI00298176
9
2.945
9
LOC442497; SLC3A2 solute
IPI00554722
13
2.9
12
carrier family 3 (activators
of dibasic and neutral amino
acid transport), member
2 isoform e
SAE1 SUMO-activating enzyme
IPI00033130
8
2.89
9
subunit 1
IPO7 Importin-7
IPI00007402
11
2.85
9
SET Isoform 2 of Protein SET
IPI00301311
9
2.85
11
PAICS Multifunctional protein
IPI00217223
15
2.84
11
ADE2
OCIAD2 Isoform 1 of OCIA
IPI00555902
6
2.83
10
domain-containing protein 2
GLT25D1 Glycosyltransferase 25
IPI00168262
5
2.82
7
family member 1 precursor
RCC2 Protein RCC2
IPI00465044
12
2.79
10
CTSG Cathepsin G precursor
IPI00028064
10
2.77
12
CHD4 Isoform 2 of
IPI00455210
10
2.765
8
Chromodomain-helicase-
DNA-binding protein 4
SSBP1 Single-stranded DNA-
IPI00029744
6
2.765
10
binding protein, mitochondrial
precursor
ACOT7 Isoform 1 of Cytosolic
IPI00010415
6
2.76
8
acyl coenzyme A
thioester hydrolase
AK3 GTP:AMP
IPI00465256
13
2.72
9
phosphotransferase
mitochondrial
GCA Grancalcin
IPI00004524
6
2.71
9
ACIN1 Isoform 1 of Apoptotic
IPI00007334
5
2.69
9
chromatin condensation inducer
in the nucleus
TM9SF4 Isoform 2 of
IPI00885106
8
2.69
8
Transmembrane 9 superfamily
member 4 precursor
CAD Putative uncharacterized
IPI00893035
17
2.675
9
protein CAD
FASN Fatty acid synthase
IPI00026781
74
2.66
11
TJP2 Isoform A1 of Tight junction
IPI00003843
16
2.66
8
protein ZO-2
GLRX3 Glutaredoxin-3
IPI00008552
6
2.595
7
RCC1 regulator of chromosome
IPI00787306
5
2.59
7
condensation 1 isoform b
TOP1 DNA topoisomerase 1
IPI00413611
10
2.57
9
GGH Gamma-glutamyl hydrolase
IPI00023728
11
2.565
10
precursor
PUF60 Isoform 5 of Poly
IPI00856076
10
2.56
9
TPR nuclear pore complex-
IPI00742682
16
2.545
9
associated protein TPR
GMPS GMP synthase
IPI00029079
11
2.525
10
PKP3 Plakophilin-3
IPI00026952
19
2.52
10
LOC731605 similar to BCL2-
IPI00886854
7
2.515
9
associated transcription factor 1
isoform 2
SLC2A1 Solute carrier family 2,
IPI00220194
8
2.515
7
facilitated glucose transporter
member 1
C8orf55 Uncharacterized protein
IPI00171421
7
2.51
8
C8orf55 precursor
HCFC1 Uncharacterized protein
IPI00641743
7
2.51
10
HCFC1
IPI Acc. No, accession number in IPI database;
nPEP, number of identified peptides in all the samples,
MED, median of the ratio tumor/healthy from all the patients;
#>2.5, number of patients in which tumor/healthy >2.5.
The proteins identified on the basis of the peptides detected by the multidimensional chromatography technique include those listed in Table 6, and reflects the abundance of these proteins in cancerous tissues. None of the proteins listed in Table 6 were identified by the isotopic labeling technique. The proteins listed in Tables 3 and 6 appear to be specifically expressed in colorectal cancer tissue, as they were substantially undetectable in all healthy colorectal tissues obtained from the colorectal cancer patients.
TABLE 6
IPI
Acc.
No. of
Protein Name
No.
AVG
SD
tumors
ADAMDEC1 ADAM DEC1 precursor
4480
50
0
AMACR Alpha-methylacyl-CoA
847727
50
0
7
racemase
AMACR; C1QTNF3 alpha-methylacyl-
5918
50
0
7
CoA racemase isoform 1
ARID1A Isoform 1 of AT-rich interactive
643722
50
0
domain-containing protein 1A
CEBPZ CCAAT/enhancer-binding
306723
50
0
protein zeta
COL5A1 Collagen alpha-1(V) chain
844090
50
0
precursor
EFEMP2 Mutant p53 binding protein 1
556657
50
0
variant (Fragment)
FAM84B Protein FAM84B
64666
50
0
FKBP10 FK506-binding protein 10
303300
50
0
8
precursor
FKBP9 FK506-binding protein 9
182126
50
0
9
precursor
GPRC5A Retinoic acid-induced protein 3
22624
50
0
8
KPNA2 Karyopherin alpha 2
789457
50
0
MMP1 Interstitial collagenase precursor
8561
50
0
PNMA5 Paraneoplastic antigen-like
514588
50
0
protein 5
POLR1C Isoform 1 of DNA-directed
5179
50
0
9
RNA polymerases I and III subunit
RPAC1
SPARC SPARC precursor
14572
50
0
5
UBAP2 Ubiquitin-associated protein 2
171127
50
0
5
UCK2 Isoform 1 of Uridine-cytidine
65671
50
0
kinase 2
WDR74 Isoform 1 of WD repeat-
18192
50
0
containing protein 74
Table 7 lists proteins that are considered potential targets for development of cytotoxic reagents specifically directed to these proteins, for example, specific antibody or antibody fragments conjugated to toxic moieties for targeted elimination of cancer cells. As well as being highly expressed in early stage polyps and in tumors, these proteins are generally exposed, and considered vulnerable to attack by targeted cytotoxic reagents.
TABLE 7
Protein
(IPI Acc. No.)
nBigTumor
nSmallTumor
nBigPolyp
nSmallPolyp
Ontology
DEFA3 Neutrophil
26
2
5
0
EX
defensin 3 precursor
(IPI00021827)
ELA2 Leukocyte
23
0
6
0
EX
elastase precursor
(IPI00027769)
LYZ Lysozyme C
17
2
8
0
EX
precursor
(IPI00019038)
LOC442497; SLC3A2
16
1
8
0
PM
solute carrier family 3
(activators of dibasic
and neutral amino acid
transport), member 2
isoform e
(IPI00554722)
SLC1A5 Neutral
16
0
6
0
PM
amino acid transporter B
(IPI00019472)
DMBT1 Isoform 1 of
15
0
8
1
EX AM
Deleted in malignant
brain tumors 1 protein
precursor
(IPI00099110)
NUCB1 Nucleobindin-
15
3
7
0
EX AM
1 precursor
(IPI00295542)
SLC12A2 Isoform 1 of
14
2
16
0
PM
Solute carrier family
12 member 2
(IPI00022649)
GGH Gamma-
14
1
12
0
EX
glutamyl hydrolase
precursor
(IPI00023728)
AGR3 Anterior
14
4
7
0
EX
gradient protein 3
homolog precursor
(IPI00152409)
MARCKSL1
13
0
11
0
PM
MARCKS-related
protein
(IPI00641181)
TM9SF2
12
1
9
0
PM
Transmembrane 9
superfamily member 2
precursor
(IPI00018415)
SYK Isoform Long of
11
0
10
0
PM
Tyrosine-protein
kinase SYK
(IPI00018597)
GCA Grancalcin
11
0
7
0
PM
(IPI00004524)
HDLBP Vigilin
11
2
6
0
PM
(IPI00022228)
C1QBP Complement
11
2
6
0
PM
component 1 Q
subcomponent-binding
protein, mitochondrial
precursor
(IPI00014230)
KIAA0152
10
0
13
0
PM
Uncharacterized
protein KIAA0152
precursor
(IPI00029046)
CLIC1 Chloride
10
2
6
0
PM
intracellular channel
protein 1
(IPI00010896)
nBigTumor, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was >3;
nSmallTumor, number of patients in which the ratio (expression level in tumor tissue/expression level in healthy tissue) was <1/3;
nBigPolyp, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was >3;
nSmallPolyp, number of patients in which the ratio (expression level in polyp tissue/expression level in healthy tissue) was <1/3;
EX, extracellular region;
PM, plasma membrane;
AM, additional membrane.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the brand concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.
Beer, Ilan, Admon, Arie, Barnea, Eilon, Ziv, Tamar
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